2011 - Chromatography of Membrane Proteins and Ins

Chromatography of Membrane Proteins and Lipoproteins
Lello Zolla and Angelo DAlessandro University of Tuscia, Viterbo, Italy

1 Introduction 2 Chromatography of Membrane Proteins 2.1 Methods for Protein Separation and Characterization 2.2 Separation Depending on the Membrane Protein Category 3 Chromatography of Lipoproteins 3.1 Methods of Lipoprotein Separation 4 Electrophoresis of Membrane Proteins: A Brief Overview 4.1 Classical Gel-based Approaches 4.2 Native Gel-based Approaches 5 Examples of Application 5.1 Experimental Considerations 5.2 Membrane Proteins 5.3 Separation of Lipoproteins 6 Current Trends 6.1 Membrane Proteins 6.2 Lipoproteins 7 Comparison with Other Methods Acknowledgments Abbreviations and Acronyms Related Articles References 1 2 3
tool for the characterization of virtually any hydrophobic protein. Application examples are described, and comparisons with other methods are discussed. Moreover, HPLC is not a destructive technique, and therefore, proteins, once separated, are available for further analytical investigations. Among these techniques, quantitative and qualitative analyses of the separated fractions can be obtained through other biophysical approaches, such as crystallography or structural spectroscopy. Most of these approaches require preliminary protein purication (90% or higher), which could be rapidly obtained through preliminary HPLC.
1
5 7 7 9 9 9 9 10 11 23 26 26 29 29 32 32 33 34
INTRODUCTION
The available methods for the separation of membrane proteins and lipoproteins are sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS/PAGE), followed by immunoblotting, isoelectric focusing (IEF), and capillary electrophoresis (CE), along with the recently introduced gel-based native techniques (blue native (BN) and clear native (CN)), and high-performance liquid chromatography (HPLC). In this article, it is shown that HPLC techniques, given their wide versatility, relative ease of use, and high resolution, may be considered the most valuable
Update based on original article by Lello Zolla, Encyclopedia of Analytical Chemistry, 2000, John Wiley & Sons Ltd. Tel.: +39 0761 357 100; fax: +39 0761 357 630. E-mail address: zolla@unitus.it
The importance of membrane proteins is highlighted by the fact that about one-third of all the genes in various organisms code for this class of proteins.(1) Over two-thirds of all medications exert their effects through membrane proteins, which makes them a major target of pharmacological interest.(2) However, approaches targeting membrane proteins are hampered by unminor technical challenges. Owing to their lipophilic character, the solubilization and separation of membrane proteins and lipoproteins normally requires the use of detergents. Consequently, classical protein purication strategies, designed for water-soluble proteins, are of limited value, and the number of reports dealing with the separation and characterization of such proteins is limited. Moreover, their hydrophobic nature induces selfassociation into noncovalent multimers, and therefore, all separative methods require preliminary procedures such as tedious sequential gradient ultracentrifugation for sample preparation, with the risk of affecting the results. Although the available methods for the nal separation of these hydrophobic proteins are numerous, their high hydrophobicity and the presence of detergents make most of these available methods expensive and technically demanding.(3) The traditional approaches by SDS/PAGE are not only cumbersome but also rather ineffective for evaluating differences in small molecular masses, and the run times required are very long, typically more than 2030 h. More complex gelbased approaches, such as two-dimensional isoelectrofocusing/sodium dodecyl sulfate polyacrylamide gel electrophoresis (2-D-IEF/SDS-PAGE) hold a great separation potential. Although two-dimensional electophoresis (2-DE) has many benets, the technique does not lend itself to large-scale, high-throughput proteomic analyses, as not all types of proteins are well resolved in this system. Proteins bearing extremes of size, hydrophobicity, or charge fail to enter the gel and are not or underrepresented.(4) Although membrane proteins
Encyclopedia of Analytical Chemistry, Online 20062011 John Wiley & Sons, Ltd. This article is 2011 John Wiley & Sons, Ltd. This article was published in the Encyclopedia of Analytical Chemistry in 2011 by John Wiley & Sons, Ltd. DOI: 10.1002/9780470027318.a1607.pub2
2
with up to 12 transmembrane -helices have been resolved and identied by 2-DE-MS (mass spectrometry), most membrane proteins have been resistant to this approach.(3) CE could represent a powerful separation technique for lipoproteins, but the number of reports on membrane proteins is still limited. It is not surprising that HPLC techniques, given their wide versatility, relative ease of use, and high resolution, may be considered the most valuable tool for the characterization of virtually any hydrophobic protein.(5) Moreover, HPLC is not a destructive technique, and therefore, proteins, once separated, are available for other analytical investigations, such as by SDS/PAGE, or other biophysical analyses, such as crystallography or structural spectroscopy. Membrane protein structural biology is still a largely unconquered area, given that approximately 25% of all proteins are membrane proteins and yet <150 unique structures are available. Membrane proteins have proven to be difcult to study owing to their partially hydrophobic surfaces, exibility, and lack of stability, although recent technical advancements make it possible to foresee a rapid expansion of the eld in the near future.(6) The real deal in these techniques is the need for preliminary purication of protein species of interest, which is best and rapidly obtained with HPLC analyses of complex samples. In any case, before loading a sample on any HPLC system, a preseparation step for membrane proteins or lipoproteins, according to their hydrophobic characteristics, must be achieved by selective extractions. The pretreatment of these proteins facilitates subsequent separation and provides a rst guideline for the choice of detergent. It is a general rule, in fact, that when choosing the detergent for the running buffers, one should use the one with which the protein was solubilized, if possible.(7) In vitro studies such as crystallization rely on the successful solubilization or reconstitution of membrane proteins, which generally involves the careful selection of solubilizing detergents and mixed lipid/detergent systems. Methods currently available for efcient reconstitution and solubilization of membrane proteins involve the use of detergent micelles, mixed lipid/detergent micelles, and bicelles or liposomes.(8) It is also advisable to use less denaturing detergents and nonionic detergents for solubilization and likewise as an additive to the running buffers, in order to retain the biological activity of proteins. This represents one of the most challenging tasks in membrane protein research, especially for structural investigation purposes, in which retaining the stability and function of the protein during solubilization, reconstitution, and crystallization is fundamental.(8) The presence of detergent in all steps preserves the subsequent tendency toward association and aggregation and the possibility of nonspecic
PEPTIDES AND PROTEINS
interactions with the support used for chromatographic separation.(8) Although lipoproteins and membrane proteins show common features, specic protocols for the separation of each group of proteins have recently been proposed, and therefore, they are presented separately.
CHROMATOGRAPHY OF MEMBRANE PROTEINS
Methods for the separation and characterization of membrane proteins differ depending on the type of interaction of the protein with the membrane: proteins embedded in a lipid bilayer (integral membrane proteins) or proteins associated with membrane structures (peripheral). In the latter case, usually ionic interactions or hydrogen bonds are involved in the anchorage, and therefore, the protein may be removed by gentle solubilization with buffered salt solution and then analyzed. Dilute sodium hydroxide and sodium hydrogen carbonate (between pH 10 and 12), concentrated salt solutions, and complex-forming substances such as ethylenediaminetetraacetic acid (EDTA) and ethylene glycol bis(aminoethyl ether)-N ,N ,N ,N -tetraacetic acid (EGTA) are used satisfactorily for their solubilization. In the case of embedded proteins, they must rst be extracted from the membrane structures and then isolated. Membrane proteins preliminary enrichment is often performed by ultracentrifugation in sucrose gradient; lectin afnity chromatography in combination with centrifugation, silica beads, or biotinylation; and interaction with immobilized streptavidin.(9) Tandem lectin afnity could also be applied as an approach for the enrichment of membrane glycoproteins. Repeated freezing and thawing can be applied to dissolve the structures mechanically, thereby allowing most membrane-associated proteins to be removed. However, solubilization of the membrane is the most suitable approach. Solubilization of membrane proteins is carried out through the use of detergents and amphipathic molecules, consisting of a polar head group and a hydrophobic chain (or tail) and exhibiting unique properties in aqueous solutions in which they spontaneously form (generally) spherical micellar structures. Membrane proteins are frequently soluble in micelles formed by amphiphillic detergents. Detergents solubilize membrane proteins by creating a mimic of the natural lipid bilayer environment normally inhabited by the protein. Four main categories of detergents are commonly used, namely, ionic detergents, bile acid salts, nonionic detergents, and zwitterionic detergents (see the article by Seddon et al.(8) for further details). Solubilization is also achieved with different detergents, such as combinations of chloroform and methanol, which
CHROMATOGRAPHY OF MEMBRANE PROTEINS AND LIPOPROTEINS
were used to extract hydrophobic chloroplast membrane proteins and could be suitable for membrane proteins of non-plant-cells as well. Alternative aqueous two-phase systems could be used, which employ the detergent DDM (n-dodecyl -D-maltoside), Triton X-114, or PEG for the selective binding of one or more proteins of interest to one of the incompatible aqueous phases. Chaotropic reagents such as urea and guanidine hydrochloride are now used less frequently, while solubilization using detergents is still in principle more practical. Membrane proteins can also be puried into lipid/detergent micelles. Indeed, it is also advisable to use less denaturing detergents such as 3-[(3-cholamidopropyl)dimethylammonio]l-propanesulfonate (CHAPS) or nonionic detergents such as n-octyl -D-glucopyranoside (OD) or n-dodecyl--Dmaltoside (-DM) for solubilization.(8) Identication of membrane proteins could be further complicated by the lack of tryptic cleavage sites across the transmembrane chain fragments. Enzymatic digestion often results in large, hydrophobic species, making it difcult to nally identify the protein species. Analyses of proteins from a number of proteomic studies of cell membranes have demonstrated that a signicant component of the identied proteins is not predicted to contain transmembrane regions. However, the presence of such proteins may sometimes arise as a result of contamination of the membrane preparations or through real associations.(10) Through the use of various reagents in different steps, the membrane proteins can be prepared according to their respective solubility and hydrophobic characteristics. The effort necessary for solubilization of the proteins increases with growing complexity of the membrane structure. Section 5.2.3 reports a complete solubilization scheme for the thylakoid membrane of chloroplasts, which contains the photosynthetic apparatus consisting of a large number of proteins, namely, 40 different proteins assembled into two main complexes: photosystem I (PSI) and photosystem II (PSII). It is shown that by selective extraction, a preseparation of membrane proteins according to their hydrophobic characteristics is achieved. This in turn allows further separation by use of different, mainly chromatographic and electrophoretic, methods. 2.1 Methods for Protein Separation and Characterization In membrane protein investigations, several chromatographic methods may be used. Generally speaking, these methods are reversed-phase high-performance liquid chromatography (RP-HPLC), afnity chromatography, size-exclusion high-performance liquid chromatography (SE-HPLC), and ion-exchange HPLC, as well as highperformance membrane chromatography.
Two-dimensional high-performance liquid chromatography (2-D-HPLC) involving orthogonal steps of RPHPLC and strong cation-exchange high-performance liquid chromatography (SCX-HPLC) have been introduced by John Yates, III, which is known as the multidimensional protein identication technology (MudPIT).(11) Usually membrane proteins are organized as multimeric units; therefore, no single technique is likely to be sufcient for the complete characterization of different proteins. Chromatographic methods offer the possibility to combine a range of techniques in order to obtain the maximum amount of relevant information consistent with an efcient use of analytical resources. It is important, therefore, to select a battery of complementary techniques that will ensure to obtain the necessary separation. Specic considerations for selection of an appropriate battery of techniques are described in Section 5. Conventional gel permeation and ion-exchange chromatography on soft gel supports, although time consuming, are used for the preparation of large quantities (1050 mg) of pure proteins, while the preparation of smaller quantities is usually performed at low pressure (fast protein liquid chromatography (FPLC)). HPLC, usually carried out at high pressure (300400 psi), has been an essential tool for the quantitative and qualitative analyses of proteins. Its success increased when rigid supports were introduced, which are able to withstand high pressures and show chemical stability within the working range of biological separations. The separation time decreased dramatically. The rst consideration in dening an HPLC method development strategy is to establish if we need proteins with preserved biological properties or rather we need their characterization. Proteins vary widely in their physicochemical properties, and these properties can have a great impact on the conditions necessary for HPLC analysis. Solution stability can differ widely from one protein to another, and for a given protein, the solution stability can be highly dependent on pH, temperature, presence of denaturants or detergents, and other factors. Such factors must be explored as part of the separation development process in order to select appropriate conditions and an appropriate detergent, under which artifactual denaturation is minimized. Other physicochemical properties to be considered include the presence or absence of free thiol groups or bound metals and the effect of the organic solvent used in RPHPLC on the secondary or tertiary structure. Finally, the presence of detergent must be taken into account along with the possibility of nonspecic interactions with the support used for chromatographic separation. Furthermore, membrane proteins readily self-associate into noncovalent multimers. Hence, in some instances
4
one may wish to determine only covalent related substances, and it is therefore appropriate to conduct the HPLC separation using conditions that dissociate the noncovalent multimers, whereas in other instances measurement of the extent of self-association may be an objective of the separation. 2.1.1 Reversed-phase High-performance Liquid Chromatography RP-HPLC seemingly offers the best resolution over all chromatographic methods for thylakoid membranes. The advent of supports designed with reduced hydrophobicity, such as the C4 column, where proteins interact lightly with the matrix, have allowed for more gentle elution conditions, with a consequently higher recovery of proteins when applied to the separation of the photosynthetic apparatus. Clearly, using organic solvents and acids would cause the biological activity of the protein to be lost. Thus, RP-HPLC is a powerful but gentle method for the isolation of membrane proteins, where the main aim is their identication or the determination of their primary structure. RP-HPLC offers the best resolution of all chromatographic methods, although up to now it has been rarely used for the separation of membrane proteins. This is because the membrane proteins can be recovered only partly and sometimes not at all under the separation conditions usually required in RP-HPLC (elution with acetonitrile or 2-propanol gradient, in the presence of 0.1% (v/v) triuoroacetic acid (TFA)). However, membrane proteins are not easy to analyze by electrospray ionization mass spectrometry (ESI-MS) because of the presence of detergents, which have to be added to the sample in order to improve protein solubilization; therefore, detergents have to be removed for successful ESI-MS analysis since they are known to inhibit ionization during the electrospray process. One way to circumvent the problems of solubility and ion suppression is the on-line hyphenation of RP-HPLC to ESI-MS, which not only efciently removes the detergent from the samples but also fractionates the various protein components before their ESI-MS investigation. Recent studies have demonstrated that monolithic capillary columns based on polystyrene/divinylbenzene (PS-DVB) allow proteins to be separated with very high separation efciency.(12) These columns are particularly suited to study membrane proteins using a unied analytical platform (see later). Despite the considerable arsenal of available technology, analytical protocols for membrane proteins vary greatly, and considerable effort is expended in customizing these protocols for the investigation of specic problems, which is why the use of microbore HPLC and packed capillary HPLC
for characterizing biosynthetic proteins is becoming increasingly widespread. 2.1.2 High-performance Afnity Chromatography Analytical afnity provides a powerful means to purify proteins and other biomolecules, with a basic two-step retentionchaotropic elution procedure with minimal nonspecic interactions, and their subsequent elution in a highly puried state and in the native state. Afnity chromatography for preparative purposes became successful when researchers improved several key features of immobilized ligand interactions with eluting macromolecules, namely, accessibility of immobilized ligand, selectivity of ligand interaction with soluble macromolecule, and reversibility of macromolecule binding, which allows their elution without denaturation. In this regard, important parameters for a successful afnity sorbent for bioseparations include mechanical, chemical, and biological stability, and also the potential for nonspecic binding. Rigid matrices such as polymeric supports, silica, and controlled pore glass (CPG) may suffer from nonspecic binding and low recovery. Cross-linked beaded agarose or cellulose offers a good compromise between mechanical stability and nonspecic binding. These matrices also have good chemical stability within the working range of biological separations, and the product recovery is excellent. The interested reader is referred to other works for a discussion of core chromatographic matrices.(13) Once a matrix has been selected, the ligand is immobilized via a stable covalent bond to avoid progressive leaching and consequent capacity loss. The most common procedure to activate agarose is the use of cyanogen bromide (CNBr), which results in the coupling of an amine group of the ligand or spacer through an isourea bond. Matrices prepared by this procedure can suffer from the serious drawback of high ligand leaching, while more stable linkages are produced by organic sulfonyl chlorides or epihalohydrins. Lectin afnity chromatography is largely used for lipoproteins, whereas two steps with protein-specic ligands, namely, immunoafnity (IA) and transitionstate ligands, are commonly used for the separation of receptors and enzyme proteins, respectively.(14) The meaning of the word afnity in the context of protein separation has undergone evolutionary changes over the years. The exploitation of molecular recognition phenomenon is no longer limited to afnity chromatography modes. Afnity-based separations today include precipitation, membrane-based purication, and twophase/three-phase extractions. Apart from the afnity ligands, which have a biological relationship (in vivo) with the target protein, a variety of other ligands are now used in the afnity-based separations. These include
dyes, chelated metal ions, and peptides obtained by phage display technology, combinatorial synthesis, ribosome display methods, and systematic evolution of ligands by exponential enrichment.(15) Proteomics also needs afnity chromatography to reduce the complexity of the system before performing analysis by electrophoresis and MS.(16) 2.1.3 Size-exclusion High-performance Liquid Chromatography The separation of membrane proteins may be achieved satisfactorily under nondenaturing conditions by SEHPLC.(17) In this case, the size of the molecules or multimeric complexes is the basis of separation, although differences of 510 kDa in molecular weight are not sufcient for a signicant separation. Since nonspecic interactions between the sample and support, and among different sample components, must be suppressed, sometimes the addition of denaturing agents, such as chaotropic reagents or sodium dodecyl sulfate (SDS), is required. Obviously, under these denaturing conditions, the resolution and yield are optimized in SE-HPLC, along with good reproducibility of the results, but proteins may be denatured. 2.1.4 Ion-exchange High-performance Liquid Chromatography Ion-exchange HPLC is based on the different ionic interactions between proteins and anionic or cationic charges of the support.(17) Unfortunately, in this type of chromatography, protein interaction with the support is usually strong and the conditions used for elution are often denaturing. The advent of supports based on agarose, which owing to its hydrophilic characteristic has a lower level of nonspecic interactions with the sample, has renewed the use of ion-exchange HPLC for protein separations. However, in the particular case of membrane proteins, although the degree of resolution of ion-exchange HPLC is surpassed only by RP-HPLC, interaction with the support is still too strong, and other methods must be chosen, or detergents must be added to the separation buffers. 2.1.5 Hydrophobic Interaction High-performance Liquid Chromatography Hydrophobic interaction HPLC can also be used for the isolation of membrane proteins, even if they are so strong that the sample can no longer be recovered from the column, even with the use of detergents or, in extreme cases, organic solvents.
2.1.6 High-performance Membrane Chromatography Red cells, biomembrane vesicles, proteoliposomes, and liposomes noncovalently immobilized in gel particles or beads have been used as stationary phases for biomembrane afnity analyses and ion-exchange chromatographic separations. Lipid monolayers coupled to silica beads have been utilized for membrane protein purication in detergent solution. Proteins are adsorbed on the liposome surfaces and subsequently separated by salt gradient elution on charged liposomes formed and entrapped in gel beads upon detergent depletion by dialysis.(18) Hence, both protein size and charge affect the separation and the elution time. These techniques, in the particular case of membrane proteins, have considerable potential because of their high resolution, short running times, and low nonspecic interactions. For more details, the reader is referred to a review.(19) 2.1.7 Multidimensional Protein Identication Technonology The archetypal approach, termed MudPIT,(20) pioneered in the laboratory of John Yates, III, has proven to be a remarkably effective and robust methodology for investigating global changes in protein expression as a function of development and disease.(21 23) In shotgun proteomics, the peptides derived from the digest of proteins are separated and analyzed in a liquid phase. This also avoids the difculty of extracting hydrophobic proteins from gels. Several shotgun approaches have been thought to circumvent the low solubility and high hydrophobicity limitations of membrane proteins by using organic solvents,(24) organic acids,(24) microwave-assisted acid hydrolysis,(25) or detergent-containing aqueous solution.(26) MudPIT couples strong cation-exchange (SCX) chromatography and reversed-phase (RP) chromatography to tandem MS in a single microcapillary column. The chromatography proceeds in cycles characterized by a progressive increase in salt concentration. This allows blowing peptides onto the SCX column, which is followed by a gradient of increasing hydrophobicity to progressively elute peptides from the RP column into the ion source. 2.2 Separation Depending on the Membrane Protein Category From a didactic point of view, proteins embedded in membranes may be divided on the basis of their biological role into receptors, glycosylated proteins, channel or carrier proteins, and structural proteins or those without a particular function. The rst consideration in dening the development strategy of an HPLC method is to establish whether
6
the main interest is isolation of the protein that is functionally active or its characterization. In the former case, nondenaturing conditions must be used, whereas in the latter case, any type of detergent or organic eluent may be used for optimum separation. 2.2.1 Cellular Receptors In the case of receptorial proteins, they rarely exist in aggregated form and are specic for recognizing a particular ligand. Thus, nondenaturating size-exclusion chromatography (SEC) may not be used, and afnity chromatography is more indicated because immobilized ligand proteins can be bound specically and eluted selectively. The natural ligand might be an ideal choice for binding selectivity but may suffer from a high intrinsic binding afnity, which could lead to inactivation of the receptor or ligand because of the harsh conditions needed for release from the afnity support. Lectin afnity chromatography is largely used for glycoproteins, and IA chromatography is the most widely used method for receptors. A more practical ligand may be a monoclonal antibody (MAb) to the receptor, which can be preselected for modest afnity and appropriate binding kinetics (on and off rates). In general, to prepare an IA sorbent, MAb is preferred over polyclonal antibody because the MAb can be obtained once a hybridoma clone has been isolated reproducibly and the appropriate MAb can be selected with the desired binding properties to optimize biomolecular adsorption and elution. The desired elution conditions can be incorporated into the screening procedure to identify the most advantageous MAb. Since the binding constant varies from clone to clone, selection of a clone producing an MAb with a desirable binding constant is necessary. An antibody that gives a good response in either Western blot or enzymelinked immunosorbent assay (ELISA) is not necessarily the best ligand for the IA sorbent. An example of this procedure is described in Section 5.2.2. Interaction analysis using such current tools as optical biosensors can be used to screen for those MAbs with a good balance of sufciently high afnity and nite off rate (see Section 6). Many receptors have been separated by IA.(27) A milestone in this eld is the separation of transferrin receptor from plasma membranes of various mammalian cells.(28) It is known that this protein binds with high afnity to immobilized diferric transferrin, whereas its afnity for apotransferrin is low. Consequently, the complex of transferrin and transferrin receptor can be dissociated by chelation of ferric ions after the addition of chelating reagents. In this way, transferrin receptor can be eluted from the column under mild conditions.
2.2.2 Carriers or Channel Proteins Translocating Ions Membrane proteins with the function of carriers or containing channels translocating ions across membranes may be separated and identied by the use of the perfusion planar lipid membrane (BLM) technique coupled with an HPLC system. This technique has been demonstrated to be efcient for the fast identication, isolation, and characterization of transport proteins (porins) in the outer membrane of bacteria and other organisms,(29) but it seems to be of general validity.(30) The method is based on solubilization of the membrane, separation of solubilized membrane proteins into fractions (100150) eluted from an HPLC column, followed by immediate screening on the BLM for channelforming activity, allowing precise localization of the porin-containing protein peak. The principle of the BLM method is simple. A planar lipid membrane interposed between two electrodes is predictably dull, showing little permeability to ions, no voltage dependence, and no interesting transport behavior. However, the introduction of a channel-forming protein, previously separated by HPLC, that is spontaneously incorporated into the bilayer dramatically alters the situation, allowing chemical substances to cross the lipid bilayer and giving rise to an ionic current. Furthermore, the opening and closing of this single ionic channel can be easily detected, and modication of the channel properties by voltage, pH, ionic composition, blockers, mutation, and chemical reagents can also be quantied. Although the BLM is an in vitro system, its validity has been demonstrated repeatedly through the more recent technique of patch clamping, giving condence that what is measured using BLM corresponds qualitatively and often quantitatively to what is observed in whole cells. BLMs have been intensively used in the identication and reconstruction of channel-forming proteins,(31) to determine the deposition of amyloid proteins observed in Alzheimers disease pathology,(32) and in the study of membrane proteins of other disease-related bacteria, whose porins or pathogenic toxins might be labile.(30,31) 2.2.3 Glycoproteins More than half of all human proteins are glycosylated. Glycosylation denes the adhesive properties of glycoconjugates, and it is largely through glycanprotein interactions that cellcell and cellpathogen contacts occur. Not surprisingly, considering the central role they play in molecular encounters, glycoprotein- and carbohydrate-based drugs and therapeutics represent a more than $20 billion market. Glycomics, the study of glycan expression in biological systems, relies on effective analytical techniques for the correlation of glycan
structure with function.(33) Determination of the carbohydrate composition, type, and branching pattern is an important step in understanding the biological function of glycoproteins and in the development of a recombinant DNA-derived glycoprotein as a pharmaceutical. Unfortunately, glycoproteins exist in a variety of biologically active forms owing to the nature of the diversity of monosaccharides and the variety of possible linkages. It may be estimated that the linkage possibilities of a hexasaccharide yield a possible 4.76 109 structures. The complexity of carbohydrate structures mandates that a variety of analytical methods be used for the study of these forms. Lectin afnity chromatography is used to separate membrane glycoproteins, but a promising new procedure is described in Section 5, which uses HPLC in conjunction with ESI-MS as a tool to identify the sites of glycosylation and the general nature of the glycosylation. ESI-MS can detect whether an oligosaccharide is O- or N-linked and differentiate between complex, high-mannose, and hybrid forms. MS analysis of glycopeptides is made challenging by the differing chemical properties of glycans and peptides. For example, although the -O-GlcNAc modication occurs to the Ser/Thr residues of many nuclear and cytoplasmic proteins, it was not detected until fairly recently because it is both uncharged and labile.(33) Other than by lectin afnity chromatography, glycopeptides may also be isolated by hydrophilic interaction solid-phase extraction or chromatography and graphitized carbon solid-phase extraction. They may also be enriched based on their high molecular weight using SEC (for review see the article by Mirzabekov et al.(31) ). 2.2.4 Others When the experimental goal does not include the analysis of the functional role of the membrane proteins and only their identication and characterization, RP-HPLC offers the best resolution of all chromatographic methods, especially when coupled on-line with a mass spectrometer. In Sections 5 and 6, some aspects of this application are presented and discussed.
triglycerides, phospholipids, and apoproteins in varying proportions. Lipoproteins have a globular structure containing hydrophobic lipids such as triglycerides and cholesterol esters within the interior core and a periphery containing more polar lipids, phospholipids, cholesterol, and proteins. Owing to their strong heterogeneity, classication of lipoproteins remains undened. Hydrated density is the more common form of lipoprotein classication. Thus, lipoproteins are separated into very low-density lipoprotein (VLDL), low-density lipoprotein (LDL), and high-density lipoprotein (HDL) fractions. In this case, lipoproteins are viewed as particles that are heterogeneous with respect to their physical properties but homogeneous with respect to apoliprotein composition. The HDL, LDL, and VLDL groups show different functions, which results in their having different properties with respect to atherosclerosis, and therefore it is very important to have methods available for their separation.(34) On the other hand, the separation and characterization of lipoproteins with respect to protein composition is of increasing interest because of the growing evidence that apolipoproteins are better markers of coronary heart disease (CHD) than serum cholesterol levels(35) and that the apoliprotein distribution is important in diagnosing lipoprotein abnormalities. Table 1 summarizes the physical and chemical properties of the main lipoprotein classes and their subfractions and also enlists the different types of apoproteins and their isomers. 3.1 Methods of Lipoprotein Separation As an alternative to ultracentrifuge separation, gel permeation separation does not require expensive ultracentrifugation instrumentation and has the advantage of being relatively mild and nondestructive in the separation of lipoprotein particles. Gel permeation chromatography (GPC) and afnity chromatography are used satisfactorily for preparative purposes and to separate lipoproteins according to their size and apolipoprotein content, respectively. However, these chromatographic methods have not gained wide acceptance as routine techniques, requiring long analysis times and excessive dilution of samples during separation. In this respect, the development of rigid supports for gel permeation has led to large improvements in speed and resolution. GPC of whole plasma is now used primarily for analytical rather than preparative separation of plasma lipoproteins. The advent of RP-HPLC resulted in renewed interest in the chromatographic separation of lipoproteins, offering the best performance in terms of speed and resolution of structural variants. In particular, the use of rigid supports, which are able to withstand high pressures, decreased the separation time dramatically to 1 h compared with 20 h by
CHROMATOGRAPHY OF LIPOPROTEINS
The term lipoprotein refers to particles that are heterogeneous with respect to size, hydrated density, and composition. The main function of the lipoprotein system is to transport lipids to the surrounding tissues. All are made up of cholesterol, cholesterol esters,
Table 1 Physical and chemical characteristics of lipoproteins: subfraction classes and apolipoproteins
Lipoprotein class VLDL LDL HDL Diameter (nm) 3070 1726 79 Subfractions 5 5 Major apolipoprotein apoC, apoB, apoE apoB, apoF apoA-I, apoA-II, apoE Apolipoprotein isomers apoC-I, C-II and C-III(0,1,2) , C-IV apoE-1, E-2, E-3, E-4, E-5 ApoD apoB100 apoB48 apoA-I1,2,3,4 apoA-II and A-IV
the conventional gel permeation method. Furthermore, the separation may be easily automated, allowing unattended analysis of multiple samples. Automation also allows on-line monitoring of cholesterol in the separated LDL and HDL fractions (see later). Two new emergent chromatographic techniques for lipoprotein separations are hydroxyapatite chromatography and countercurrent chromatography (CCC).(36) Their combined use allows a better separation and analysis of HDLs, LDLs, and VLDLs from human serum without prior ultracentrifugation (Section 6). However, ultracentrifugation is one of the eligible strategies for lipoprotein separation, often in duplicate steps.(37) 3.1.1 Separation by Particle Size and Subfractions Gel permeation is normally used to separate lipoproteins on the basis of differences in particle size. It was initially performed on cross-linked dextran supports, which yielded poor separations owing to their relatively small pore size; conversely, agarose gel, which has a larger pore size, has been found to yield better separations.(38) Usually, the eluting lipoproteins are derivatized postcolumn with an enzymatic cholesterol reagent, and therefore, the mixture is passed through a reaction capillary and detected at 500 nm. Complete separation is usually achieved in <80 min.(39) Separation of the main classes of lipoproteins into their subfractions is of growing interest since each subfraction has a different specic physiological function. By GPC, HDL particles may be divided into ve subfractions,(40) whereas by heparin afnity chromatography, HDL may be subfractionated according to the apoE content.(41,42) Five different fractions of oxidatively modied LDL have been identied through ion-exchange chromatography.(43) The human serum HDL fraction may be divided into ve or six subclasses by using hydroxyapatite chromatography.(36) 3.1.2 Separation of Apolipoprotein and Isomers Afnity chromatography allows separating lipoproteins on the basis of their apolipoprotein content through
the use of specic and reversible interactions between lipoprotein and bound ligands. Afnity chromatography of lipoproteins can be performed using groupspecic or biospecic ligands. Group-specic ligands include concanavalin A, which binds to apoB-containing lipoproteins,(44) and heparin, which interacts specifically with apoB and apoE,(45) while antibodies to specic apolipoproteins serve as biospecic ligands in IA chromatography. This technique provides the highest specicity for the separation and isolation of lipoproteins on the basis of their apolipoprotein content, while group-specic ligands are more economically convenient, but the separation is less accurate. In addition to afnity chromatography, the highperformance gel permeation, ion-exchange chromatography, or RP mode may be used to separate apolipoproteins into their isomers. Unfortunately, in these HPLC systems, only small amounts of sample (<5 mg) can be applied to the column. However, the speed (<60 min) and ease of operation make these techniques ideal for the analytical separation and characterization of apolipoproteins. GPC is often used as a preparative technique for isolating apolipoproteins derived from HDL or VLDL, while separation of isoforms can be achieved by either ion-exchange chromatography or RPHPLC. Apolipoproteins from HDL and VLDL can be separated in <60 min on high-performance TSK gel permeation columns.(46) Separations of apolipoproteins by ion-exchange chromatography are based on differences in charge. Therefore, this mode of chromatography can separate apolipoproteins with different isoelectric points.(42) In contrast, differences in hydrophobicity are the basis of separation by RP-HPLC. The RP columns usually have a lower sample capacity (<100 mg) than those used in gel permeation HPLC. Moreover, the column lifetime is relatively short owing to the instability of silica-based stationary phases at pH extremes. However, there is growing interest in the use of RP-HPLC as a rapid and highly efcient technique for monitoring heterogenicity in apolipoprotein structure.(46)
ELECTROPHORESIS OF MEMBRANE PROTEINS: A BRIEF OVERVIEW
IEF gel strips.(53) As a good alternative for 16-BAC, the compound CTAB can be used, resulting in a similar improvement in membrane protein recovery.(54) 4.2 Native Gel-based Approaches One of the eligible gel-based approaches to investigate protein complexes is BN-PAGE, which has been introduced by Schagger and Von Jagow almost 20 years ago.(55) Before performing BN-PAGE, protein complexes are usually solubilized in digitonin, DDM, or Triton-X100, and Coomassie brilliant blue G-250 is applied to provide the protein complex with a negative charge to enhance electrophoretic mobility. Typically, a native acrylamide gradient gel is used in the rst dimension to separate the complexes based on overall size. These complexes can subsequently be separated through a second-dimension SDS-PAGE on denaturation of the protein complexes. It has been shown that mitochondrial supercomplexes with a size of up to 5 MDa can be separated in this fashion.(56,57) This strategy allows separation of protein complexes and identication of interacting partners as an alternative to immune coprecipitation.(58) The advantage is that no antibodies are needed to perform this analysis, although detergentlabile interacting proteins are lost from the complexes.(59) Application of 2-D-BN/SDS-PAGE to the platelet cytosolic and membrane proteome allowed the identication of 63 proteins from different complexes, 9 of which were identied for the rst time in human platelets.(60) CyDye urophores could be used to stain the BNPAGE gels in order to perform quantitative analyses on the resolved protein complexes in a manner similar to the BN-PAGE analyses on erythrocyte membrane performed by van Gestel et al.(61) Membrane protein complexes of erythrocytes from healthy donors and patients with hemolytic anemia were compared as to individuate protein biomarkers (in this case, spectrin) to be related with membrane disorders. Unfortunately, Coomassie G-250 interferes with CyDyes. Therefore, clear native polyacrylamide gel electrophoresis (CN-PAGE) can be also performed in the absence of Coomassie G-250 (for proteins with pI < 7), while at the expenses of the capacity to resolve protein complexes as they differentially migrate merely based on their varying intrinsic charge. Mild anionic detergents, such as sodium oxycholate, have been used to cope with this inconvenience.(62)
New techniques have been recently introduced to tackle the major issues affecting classic 2-D-PAGE approaches. This mainly concerns the study of major hydrophobic proteins, namely, membrane proteins, and protein complexes through the use of native gelbased approaches. Proteins rarely function in isolation. Rather, they are organized in functional units that are different in size, the number of interacting partners, and stability, ranging from huge stable ribosomes or nuclear pore domains to small and transient signal transduction complexes, such as in the case of the band 3 interacting partners in the inner layer of red blood cell membranes, which involve multiprotein components regulating metabolic modulation through oxygensaturation-dependent reversible binding of glycolytic enzymes.(47) Membrane complexes represent physiological regulatory nuclei in anucleated cells, such as red blood cells, which are also devoid of other organelles.(48) Studying these multiprotein complexes and microdomains provides information about the spatiotemporal organization of signal transduction or metabolic processes within a cell. However, a major part of this information is lost when cells are lysed and proteins digested before analysis. Because isolated protein complexes have much reduced complexity, preliminary isolation of these complexes allows the identication of low copy number proteins from the complex and connecting them to a particular function.(49) Multiprotein complexes and associated proteins can be isolated and puried by a variety of techniques such as afnity-based, recombinant pull-downs, liquid chromatography (LC), blue native polyacrylamide gel electrophoresis (BN-PAGE), 2DE/LC/CE, and FFE (free ow electrophoresis) methods, followed by MS analysis.(50 52) 4.1 Classical Gel-based Approaches Classical gel-based approaches, such as IEF, SDS-PAGE, 16-BAC-PAGE, and CTAB-PAGE (cetyltrimethylammonium bromide polyacrylamide gel electrophoresis), are excluded, as they separate proteins under denaturing conditions.(53) These methods could be exploited to perform analyses on hydrophobic protein fractions. Indeed, the use of 16-BAC instead of SDS, which is an anionic detergent, is benecial when performing analyses on membrane proteins, as they are usually characterized by an alkalic pI. Therefore, solubilization steps with cationic detergents, such as 16-BAC, dramatically improve separation resolution during electrophoretic separation. This methodology has been shown to work well both in combination with acrylamide gels and with
EXAMPLES OF APPLICATION
As mentioned earlier, the separation of membrane proteins and lipoproteins is presented separately owing to the specicity of the methods set up for the two
10
main protein categories. However, in both cases, HPLC is one of the most convenient methods to be used, and therefore, a few considerations specically relevant to the instrumentation required are presented. 5.1 Experimental Considerations In the case of proteins, more than for small organic compounds, HPLC requires extremely precise control of the solvent composition because the RP-HPLC elution time is much more dependent on organic solvent composition. Single-pump, low-pressure mixing HPLC gradient systems are widely used for protein analysis. However, when using solvents that are difcult to mix (e.g. 1-propanol), dual-pump, high-pressure mixing is advantageous because the two solvent streams are delivered continuously rather than in a segmented manner. Moreover, with single-pump operation, the continuous sparging of solvents with helium, for degassing purposes, results in changes to the organic solvent composition. This problem may be minimized by presaturation of the helium with organic vapor by passing the sparge gas through a solvent reservoir containing the mobile phase. With respect to detection, generally either 280- or 214nm wavelength is employed in protein analysis. Detection at 214 nm is more sensitive and more generally applicable because absorption at this wavelength is due to the peptide backbone. However, some HPLC mobile-phase constituents may interfere at this wavelength (e.g. acetate salts). Detection at 280 nm is somewhat less sensitive but can be used to detect most proteins (absorption at 280 nm is primarily due to the aromatic amino acid side chains of tryptophan, phenylalanine, and tyrosine). In some cases, dual-wavelength or diode-array detectors are recommended in order to provide more detailed spectral information for each peak observed. In general, the sample solution should be similar in composition (e.g. pH) to the HPLC mobile phase so as to minimize the effect of the sample injection on the separation efciency. One exception to this rule is RP-HPLC, where the organic solvent composition of the sample should be less than that of the initial mobile phase in order to focus the injected proteins at the head of the column. Clearly, the protein must have adequate stability in the selected solvent, and in many cases, stability must be enhanced by use of a refrigerated autoinjector. When conducting SE-HPLC for determining noncovalent multimers, one must avoid the use of denaturants (e.g. organic solvents) or extreme pH conditions that might disturb the equilibrium between monomeric and multimeric forms.(17) The column is the heart of the HPLC separation system. Unfortunately, this is the one component over
which the analyst has the least direct control, exception made for direct control of the temperature of the column. Consequently, it is important to work closely with the column manufacturer to obtain several independent column-manufacturing batches and to investigate whether a particular method performs consistently on a number of such batches. Whenever possible, an HPLC method to be used on a routine basis should be validated using two alternative sources (brands) of columns in order to minimize the impact of column-manufacture problems. The advent of new RPs such as C-4 allowed excellent separations of hydrophobic proteins with a high protein recovery (see later). Upon approximately 20 years of stagnation, packed-column technology has begun to evolve rapidly in the past 10 years. The rst monolithic columns appeared at the turn of the last century, threatening the monopoly of columns packed with particles but slowly losing momentum. Then, the average particle size routinely used in commercial columns began to decrease progressively, rst from 5 to 3 m and lately to sub-2-m particles. Columns packed with particles of the latter particle size allowed reaching high-resolution power with reduced plate heights as low as 3.2 m (i.e. Ca 300 000 plates per meter) for small molecules and reduced analysis times by Ca one order of magnitude. The main disadvantage of such columns is the need for suitable instruments that are capable of operating at a pressure as high as 15 000 psi (1000 bar) and to record without signicant distortion to the very narrow peaks eluted from the columns. In about the same time when sub-2-m particles were commercialized, other scientists searched to prepare particles that are large enough to be used at velocities somewhat larger than their optimum velocity for maximum efciency and exhibit low mass transfer resistance. This combination can provide columns that can be operated with conventional pumps but have efciency comparable to that of columns packed with sub-2-m particles. Nonetheless, C4 and C8 columns can often be useful where separations are difcult with C18 columns. The use of microbore HPLC and packed capillary HPLC for the characterization of biosynthetic proteins has become more widespread. One of the main advantages is the lower solvent consumption. Furthermore, such techniques are more compatible with highperformance liquid chromatography/mass spectrometry (HPLC/MS) systems, which are now widely available and offer profound advantages for method development (Section 6). The majority of the protein-oriented literature describes efforts for improving resolution of the separation and increasing the sequence coverage of a protein by separating complex mixtures of tryptically digested proteins. Methods developed, such as peak parking and multidimensional separations, have
11
Unbound peak Absorbance (280 nm) Eluted peak
increased the sensitivity of sample analysis and enabled further insight into the protein sample. However, sensitivity is the driving factor to develop HPLC columns of smaller and smaller diameters and to develop new types of stationary phases, which are capable of selectively binding certain types of analyte. Theoretically, reducing the internal diameter (ID) of a column from d1 to d2 results in an enhanced sensitivity (f ), given by f = d1 2 /d2 2 . This means that a change in column ID from 300 to 75 m would result in a 16-fold increase in sensitivity or, more obviously, the change from a conventional 4.6-mm- to 75m-ID column would result in a (theoretical) sensitivity gain of more than 3600-fold.(63) Taken together, these reasons contributed to make the nano-HPLC-MS workow the strategy of choice for identication of proteins from biological mixtures, especially upon preliminary separation steps involving gel-based approaches (either 1-D-SDS-PAGE or 2-D-GE (gel electrophoresis)). 5.2 Membrane Proteins Here three examples are presented. 5.2.1 Cellular Receptor CD4 is an integral membrane glycoprotein of T cells, which acts as the cellular receptor for human immunodeciency virus (HIV). Many protocols have been set up to recognize and separate CD4. All are based on IA using MAbs and autoantibodies(64) directed against conformational epitopes of CD4. 5.2.1.1 Selecting a Monoclonal Antibody Soluble CD4 (sCD4) had been considered as a possible therapeutic agent for acquired immune deciency syndrome (AIDS) by acting as a molecular decoy, i.e. by binding to the gp120 coat protein of HIV and thereby preventing cellular binding of HIV. Consequently, there has been a growing interest in searching CD4 variants from either mammalian cell culture or microbial extracts, showing improved pharmacokinetic properties. A rapid generic purication scheme for sCD4 constructs has been developed using IA separation. To prepare a robust IA sorbent for the purication of sCD4, a number of sCD4 mutants and a series of MAbs were examined. Five different commercial anti-CD4 MAbs were immobilized with the protein amino groups on to a Sepharose matrix containing an 11-atom spacer using active ester chemistry, and all the sorbents were evaluated individually on a small test column. Figure 1 shows representative chromatograms to illustrate that different antibodies bind CD4 differently. In the cases of L-92.5 and L-83 clones, the binding was restrictively tight, whereas L-71, L-77, and L-104.5
L-71
L-83 L-92.5 Elution volume Ligand L-71 Load (g) 1400 Unbound 1062 Eluted 336 L-83 1400 1306 55 L-92.5 1400 820 61
Figure 1 Absorbance (A280 ) proles of sCD4 binding to IA column. sCD4, at 0.1 mg mL1 in N-(2-hydroxyethyl)piperazine-N-(2-ethanesulfonic acid) (HEPES) buffer at pH 7.5, was loaded at 60 cm h1 at 4 C on to a 1- 6.4-cm IA column, preequilibrated with 0.05 M HEPES, 0.15 M NaCl, 0.01% PEG 3400 (pH 7.5). To remove nonspecically bound proteins, the column buffer was washed with two column volumes of HEPES containing 0.5 M NaCl at the same ow rate. Bound sCD4 was eluted with 0.1 M acetic acid, 0.15 M NaCl, 0.01% PEG 3400 at 60 cm h1 . The inset shows the quantities of sCD4 in various fractions determined by Bio-Rad protein determination.(65) (Reproduced by permission of Elsevier Science from C. Jones, A. Patel, S. Grifn, J. Martin, P. Young, K. ODonnell, C. Silverman, T. Porter, I. Chaiken, J. Chromatogr. A, 707, 322 (1995).) showed moderate and more tractable binding afnity. Although L-92.5 sorbent bound the highest amount of CD4, the recovery was the least of all the sorbents. On the other hand, the L-71 sorbent showed moderate binding, but the recovery of sCD4 was quantitative. Thus, MAb L-71 was judged to be the most suitable candidate for immobilization to prepare an afnity sorbent to purify CD4 and a scale-up IA sorbent to purify CD4 congeners. Using this antibody as IA sorbent, recombinant proteins loaded on to the column were recovered with very high yield using elution with 0.1 M acetic acid. The proteins recovered formed an electrophoretically homogeneous product and after neutralization were highly active with respect to gp120 binding, as judged by a radioligand-bead binding assay. The sorbent was also used successfully to purify full-length CD4 in highly active form. A large-scale IA purication process was developed for the isolation of recombinant sCD4 from Escherichia coli fermentations.(66) The MAb used for IA purication of sCD4 recognized a conformation-dependent epitope on the surface of the domain 1 of CD4. IA chromatography was used to purify both sCD4183, consisting of 183 Nterminal amino acids of human CD4, and sCD4PE40, a
12
fusion protein consisting of 178 N-terminal amino acids of CD4 and amino acids 13 and 253613 of Pseudomonas exotoxin A (PE40). Relatively high recoveries of sCD4183 and sCD4PE40 were observed in the IA step of the purication process (71% and 79% recovery, respectively). sCD4183 was puried from E. coli cell pellets using cell disruption, protein solubilization, oxidation, Q-Sepharose anion-exchange chromatography, and IA chromatography steps. sCD4PE40 was puried from cell pellets using cell disruption, protein solubilization, oxidation, Cu2+ -immobilized metal-afnity chromatography, anion-exchange chromatography, and IA chromatography steps. The immobilized MAb appeared to be selective for correctly folded CD4 protein since sCD4183 and sCD4PE40 puried by the IA method bound the HIV glycoprotein gp120 (HIV gp120) in vitro. The results demonstrate that immobilized MAbs directed against conformational epitopes may be used for the rapid purication of gram amounts of correctly folded protein from mixtures of oxidized E. coli proteins. 5.2.2 Red Blood Cell Membrane Molecular masses of the proteins contained in red blood cell membrane (Figure 2) range between 20 and 200 kDa, and consequently, the most common HPLC techniques
used for the purication of these proteins are based on SEC (which may be used in the presence of either ionic or nonionic detergents) and, to a lesser extent, on ionexchange or hydrophobic interaction chromatography (compatible with nonionic detergents). We have used an RP C-4 column for the separation of membrane proteins from erythrocyte ghosts using mobile phases containing acetonitrile and 0.1% TFA. The erythrocytes were swollen in 5 mM Na2 HPO4 isotonic solution, and after several centrifugations at 3000g the pellet was solubilized in two volumes of pure acetonitrile. Samples were loaded directly on the column without any other manipulation. Figure 3 shows the chromatogram obtained using a gradient of acetonitrile. A silica-based C-4 column eluted with TFAacetonitrile is a perfect combination for several polypeptide separations but was considerably less suitable for membrane protein separation than a resin-based phenyl column eluted with the same mobile phase. In our case, the separation obtained with Vydac C-4 may be considered excellent, better than that reported previously with a Nucleosil C-4 column(67) where only one main peak was observed. In our opinion, the better separation is related to the absence of detergent during the extraction and elution run and probably also to the better quality of resin in the column.
3 GP
Aldolase Aldolase Hemichrome GAPDH PFK
GP 4.2
4.1
Hemichrome GAPDH
4.2
4.1
Ankyrin
PFK
Ankyrin
4.2
Spectrin dimers
Ankyrin
S S
Figure 2 A schematic view of the organization of the main red blood cell membrane proteins.
13
Absorbance
0.18
centrifuged (10 min, RT), and the supernatant was discarded. The whole complex proteomic workow, including SDS-PAGE, digestion of separated bands, and capillary liquid chromatography/MS analysis, yielded identication of 340 independent protein spots.(68)
0.03
0.12 0 5 10 15 20 Time (min) 25 30 35 40
5.2.4 From Extensive Basic Studies toward Clinical Application Preliminary 2-D-IEF-SDS-PAGE, trypsin digestion of spots of interest, and nano-HPLC-ESI-MS have revealed far more membrane proteins in a single approach.(69) The erythrocytes were isolated by centrifuging twice at 1000g for 10 min. Packed cells were washed three times in 5 mM phosphate buffer, pH 8.0, containing 0.9% (w/v) NaCl; then, they were centrifuged at 300g for 10 min, at 4 C. Erythrocytes were lysed with nine volumes of cold 5 mM phosphate buffer, pH 8.0, containing 1 mM EDTA and 1 mM phenylmethanesulfonyl uoride (PMSF). Membranes were collected by centrifugation at 17 000g for 20 min at 4 C and further washed until free of hemoglobin. To remove lipids, proteins were precipitated from a desired volume of each sample with a cold mix of tri-n-butyl phosphate/acetone/methanol (1:12:1). After incubation at 4 C for 90 min, the precipitate was pelleted by centrifugation at 2800g for 20 min at 4 C. Samples were therefore analyzed through 2-DIEF-PAGE. Spots of interest were carefully excised from the 2-D gel and subjected to in-gel trypsin digestion according to Shevchenko et al.(70) , with minor modications. The gel pieces were swollen in a digestion buffer containing 50 mM NH4 HCO3 and 12.5 ng/L trypsin (modied porcine trypsin, sequencing grade, Promega, Madison, WI, USA) in an ice bath. After 30 min, the supernatant was removed and discarded; then, 20 L of 50 mM NH4 HCO3 was added to the gel pieces, and digestion was allowed to proceed overnight at 37 C. The supernatant containing tryptic peptides was dried by vacuum centrifugation. Before MS analysis, the peptide mixtures were redissolved in 10 L of 5% FA (formic acid). Peptide mixtures were separated using a nanoow-HPLC system (Ultimate; Switchos; Famos; LC Packings, Amsterdam, The Netherlands). A sample volume of 10 L was loaded by the autosampler onto a homemade 2-cm fused silica precolumn (75 m ID; 375 m o.d.; Resprosil C18-AQ, 3 m (AmmerbuchEntringen, DE, Germany)) at a ow rate of 2 L min1 . Sequential elution of peptides was accomplished using a ow rate of 200 nL min1 and a linear gradient from solution A (2% acetonitrile and 0.1% FA) to 50% of solution B (98% acetonitrile and 0.1% FA) in 40 min
Figure 3 Separation of erythrocyte membrane proteins

extracted in pure acetonitrile. Inset shows SDS/PAGE of proteins loaded into the column. Vydac C-4 column (250 4.6 mm ID) eluted at 1.0 mL min1 with an acetonitrile gradient (20100%) in 0.1% TFA over 60 min.
5.2.3 Red Blood Cell Membrane Proteins Characterization Through Preliminary Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis and High-performance Liquid Chromatography/Mass Spectrometry Identication Recently, the proteomics workow including preliminary separation steps through gel-based approaches, digestion of bands/spots of interest, and identication by MS has helped dramatically expand the red blood cell membrane proteome.(68,69) Packed RBCs (10 mL) were suspended in 50-mL icecold 5 mM phosphate buffer, pH 8, and centrifuged (9000g, 20 min, 4 C). Hemolysate was discarded and the operation repeated (at least ve times) until the supernatant appeared colorless. Centrifugation was then increased to 20 000g, and washing was repeated until the ghost membranes appeared yellowish white. Membranes were stored at 80 C. Protein extraction has been performed with Na2 CO3 or EtOH. In the former case, RBC membranes (10 L; 80 g protein) were resuspended in 1 mL 100 mM Na2 CO3 (pH 11) and passed ve times through a 25-gauge needle, mixed by rotation (30 min, 4 C), and pelleted (90 min, 245 000g), and the supernatant was removed. This process of suspension, rotation, pelleting, and washing was repeated twice. The pellet was either digested directly or treated with EtOH. In the second protocol of membrane protein extraction, RBC membrane pellets were diluted with four volumes absolute EtOH and brought to 50 mM sodium acetate, using 2.5 M sodium acetate, pH 5.0. Final pH was approximately 7.5. Twenty micrograms of glycogen per mililiter of the original sample was added, the suspension was mixed at room temperature (RT) for 90 min and
14
over the precolumn in-line with a homemade 10- to 15cm resolving column (75 m ID; 375 m o.d.; Resprosil C18-AQ, 3 m (Ammerbuch-Entringen, Germany)). Peptides were eluted directly into a high-capacity ion trap (model HCTplus Bruker-Daltonik, Germany). Capillary voltage was 1.52 kV, and a dry gas ow rate of 10 L min1 was used with a temperature of 200 C. The scan range used was from 300 to 1800 m/z. Although time consuming, this workow allowed individuation of protein fragments and aggregates emerging upon prolonged storage of erythrocyte concentrates under blood-banking conditions. These evidences revealed that either structural or functional proteins suffer from irreversible damage as storage progresses. Among these proteins, notable was the presence of band 3, bands 4.1 and 4.2, glyceraldheyde-3-phosphate dehydrogenase, and ankyrin, which are either structurally or functionally involved in erythrocyte survival in vitro and in vivo. 5.2.5 Separation of Photosynthetic Proteins from the Thylakoid Membrane of Chloroplasts In this section, we present the results of a study performed to develop a rapid and straightforward HPLC method to resolve and identify the protein components of PSI and PSII. These two large complexes, of at least 40 proteins, are present in the thylakoid membrane of chloroplasts (Figure 4) and have the main role of capturing the light and giving the necessary energy to transfer electrons from water to nicotinamide adenine dinucleotide phosphate (NADP) via the two photosystems, PSI and PSII, and a number of electron carriers.
PSII is embedded in the thylakoid membrane and contains a reaction center (core) of 21 large and smaller proteins, surrounded by a specic light-harvesting system, which is the major proteinchlorophyll a b complex (light-harvesting complex of photosystem II (LHCII)) and minor antennas called CP29, CP26, and CP24.(72) In contrast, structural information on PSI at the atomic level is lacking. The proteins associated with PSI and PSII have been traditionally resolved by SDS/PAGE into several closely related hydrophobic membrane proteins, typically in the range of 2140 kDa.(73) The uncertainty of the number of proteins in the complex is mainly due to the different procedures and detergents used to solubilize PSI and PSII from the thylakoid membranes and the different methods employed in isolating and characterizing the individual membrane proteins in the complex. The antenna proteins show amino acid compositions similar in length and sequence,(74) and therefore, the characterization of these proteins is performed by tedious electrophoresis and immunoblotting, which require run times of 2030 h.(75) Traditional approaches by SDS/PAGE are not only cumbersome but also rather ineffective for evaluating differences in the relative quantity of each LHCII component. We have set up a high-performance separation technique for the resolution of thylakoid membrane proteins, employing an RP Vydac Protein C-4 column containing 5-m pores.(76) 5.2.5.1 Preparation of Photosystem I and Photosystem II In Figure 5 are summarized the main steps to separate the PSI and PSII present in the thylakoid membrane.
Carbon fixing reactions
3H ATP b g a d Em
oscp
h hn H
Lhcb 1+2+3 Lhcb 4 Lhcb 5 Lhcb 6
NADP+
ADP + Pi
H Fd M G e- E L I DF F C H J K B A Lhca 1-4 P700 e N PC F PC Fx A A Lhca 1-4
CF1
Stroma
I J KW
C(CP43)
B(CP47)
(D2)
QA Phe
S N L MX H 1 PQH QB 2 e 2 A
(D1)
b6 FeS IV Q cyt b cyt b e QH e FeS cytf
I II
IV CH0 III
E F Membrane P680 Y e Y
Mn Mn Mn Mn
PQ pool
Lumen
(CP43) 1 2 H2 O
P Q
O R
T
(CP47) 1 O + 4 2
H H
Fe
PSII
Cyt b6f
PSI
ATP synthase
Figure 4 Organization of proteins contained in the thylakoid membrane and involved in photosynthesis.(71) (Reproduced by
permission of Professor J. Barber, Imperial College, London.)
15
Leaf
Freezing at 80 C and homogenization Centrifugation at 1400g for 10 min
Chloroplast
Centrifugation at 10 000g for 10 min
Thylakoids
Ultracentrifugation at 40 000g for 30 min
Solubilization in 1.5% -DM for 30 min Ion exchange by DEAE + Size exclusion by ultragel AcA34(36)
Pellet PSII
Supernatant PSI + PSII
Solubilization in 1% -DM for 20 min Ultracentrifugation at 39 000 rpm for 18 h in 01M sucrose gradient
Concentrated and solubilized in 1% -DM for 20 min Ultracentrifugation at 28 000 rpm for 42 h in 01M sucrose gradient
1 2 3 4 5 1 2 3 4 5 PSII PSI
PSI
41 000 rpm for 24 h
Figure 5 Separation scheme for PSII and PSI. Chloroplasts were isolated from spinach leaves according to the method of Berthold et al.(77) with minor modications. Leaves were powdered in liquid nitrogen and subsequently homogenized in an ice-cold 20 mM tricine buffer (pH 7.8) containing 0.3 M sucrose and 5.0 mM magnesium chloride (buffer B1). The homogenization was followed by ltration through one layer of Miracloth (Calbiochem, San Diego, CA, USA) and
16
centrifugation at 4000g for 10 min at 4 C. Pellets were suspended in buffer B1 and centrifuged as above. These second pellets were resuspended in 20 mM tricine buffer (pH 7.8) containing 70 mM sucrose and 5.0 mM magnesium chloride (buffer B2) and centrifuged at 4500g for 10 min. Pellets containing the thylakoid membranes were then resuspended in 50 mM morpholinoethanesulfonic acid (MES) buffer (pH 6.3) containing 15 mM sodium chloride and 5 mM magnesium chloride (buffer B3) at 2.0 mg mL1 chlorophyll for 15 min after adding Triton X-100 at a nal ratio of 20 mg mg1 chlorophyll. The concentration of chlorophyll was determined according to the method described by Porra et al.(78) The incubation was terminated by centrifugation at 40 000g for 30 min at 4 C. This pellet containing the PSII complex and corresponding to the BBY preparation described by Berthold et al.(77) was resuspended in buffer B3 containing 20% (v/v) glycerol and stored at 80 C. 5.2.5.2 Subfractionation of Photosystem II by Sucrose Gradient Ultracentrifugation Once isolated from thylakoid membranes, PSII membranes were subjected to sucrose gradient ultracentrifugation in order to isolate the protein components of the minor antenna system (band 2) from the major (band 3) and also from the reaction-center complexes (bands 45). To this end, PSII membranes were pelleted by centrifugation at 10 000g for 5.0 min at 4 C, suspended in buffer B3 at 1.0 mg mL1 chlorophyll, and then solubilized by adding 1% (w/v) -DM. Unsolubilized material was removed by centrifugation at 10 000g for 10 min. The supernatant was rapidly loaded on to a 0.11.0 M sucrose gradient containing buffer B3 and 5.0 mM -DM. The gradient was then spun on a Kontron (Milan, Italy) Centricon T-1080 ultracentrifuge equipped with a TST 41.14 rotor at 39 000 rpm for 18 h at 4 C. Green bands were harvested with a syringe. The SDS/PAGE analysis of these green bands revealed that band 3 contained essentially LHCII proteins, as reported previously by Bassi and Dainese.(79) The puried material was subsequently used for the HPLC study. 5.2.5.3 Separation of Photosystem II Proteins by Reversed-phase High-performance Liquid Chromatography In this study,(80) we searched for the conditions to resolve the protein components of the major and the minor antenna system of PSII either as the complex isolated by sucrose gradient ultracentrifugation or as assembled in the grana membrane (BBY particles), using an analytical (250 4.6 mm ID) or a semipreparative (250 10 mm ID) sized column, both packed with the same 5-m spherical Vydac C-4 stationary phase. The use of a semipreparative sized column was needed in order to obtain a sufcient amount of puried polypeptide in order to perform peak identication by SDS/PAGE,
immunoblotting, and amino acid microsequencing. The optimum separation of the protein components of the PSII antenna system was obtained by the following procedure. The Vydac C-4 columns were preequilibrated with 38% (v/v) aqueous acetonitrile solution containing 0.1% (v/v) TFA, and samples were eluted using either gradient I or II, depending on the HPLC unit employed for the separation. Gradient I consisted of a rst linear gradient from 38% to 55.4% (v/v) acetonitrile in 22 min, followed by 3 min of isocratic elution with the eluent containing 55.4% acetonitrile, a second gradient segment from 55.4% to 61.8% (v/v) acetonitrile in 8 min, and then a third gradient segment from 61.8% to 95% acetonitrile in 1 min. Gradient II consisted of a rst linear gradient from 38.0% to 61.8% (v/v) acetonitrile in 40 min, followed by a second gradient segment from 61.8% to 95% (v/v) acetonitrile in 1 min. For both gradients, the last gradient segment up to 95% acetonitrile was used for washing out hydrophobic contaminants of the PSII antenna system from the column. Gradient I was used to elute either the analytical or the semipreparative column. The ow rate was 1.0 mL min1 with the analytical column and 4.7 mL min1 with the semipreparative column. These conditions were selected in order to maintain the same gradient shape with both columns by keeping the ratio of the gradient volume to the column volume constant. The chromatogram displayed in Figure 6(a) shows that the material harvested from the second band of the sucrose gradient ultracentrifugation was resolved into eight main peaks and contained a mixture of the protein components of both the major and the minor PSII antenna systems. Five of these peaks, with corresponding retention times, were also obtained on separating, under the same conditions, the material harvested from the third band of the sucrose gradient ultracentrifugation (Figure 6b), which essentially contained the protein components of the major PSII antenna system. From these data, it can be inferred that the peaks labeled 1, 2, 5, 6, and 7 correspond to components present in both the sucrose bands 2 and 3, whereas peaks 3, 4, and 8 are essentially due to the component present only in the sucrose band 2, which can be tentatively identied with the minor antenna complexes CP29, CP26, and CP24.(80) Figure 6(c) shows the chromatogram of BBY injected on the column directly, avoiding the separation step by sucrose gradient ultracentrifugation. It can be observed that on injection of BBY directly, the protein components of the PSII major and minor antenna system are well resolved without interference from the other protein components of PSII. Therefore, the use of the crude PSII membrane preparation does not affect the resolution and retention times, while new and smaller peaks appear at longer elution times. The new peaks observed in Figure 6(c)

56 4 3 0.015 2 7 8
17
1 Absorbance
0.005 0 (a) 0.035 Absorbance 0.025 0.015 0.005 0.005 0 (b) 10 20 Time (min) 30 40 10 20 Time (min) 30 40
0.58 Absorbance
0.28
0.02 0 (c) 10 20 Time (min) 30 40
Figure 6 RP-HPLC separation of spinach chlorophyll a/b binding proteins contained in (a) band 2 and (b) band 3 of spinach BBY fractionated by sucrose gradient ultracentrifugation and (c) in whole BBY, directly injected into the column. Column, Vydac Protein C-4 (250 10 mm ID), eluted with an increasing acetonitrile concentration gradient as described in the text. Flow rate, 4.7 mL min1 ; detection at 214 nm. represent the core proteins: being more hydrophobic, they tend to elute at higher concentrations of acetonitrile (data not shown). In order to assign a name to each HPLC peak of the well-known antenna proteins, SDS/PAGE employing different buffer systems and either Coomassie brilliant blue or silver staining followed by immunoblotting detection with antisera directed to the individual antenna proteins were performed, in addition to amino acid microsequence analysis of the material collected throughout the chromatogram. In the case of SDS/PAGE and immunoblotting, each fraction collected from the semipreparative chromatographic separation lyophilized were dissolved in 120 mM tris(hydroxymethyl)aminomethane (TRIS)-HCl buffer (pH 8.45), containing 120 mM dithiothreitol (DTT), 5 M urea, and 4% (w/v) SDS, and then analyzed
by SDS/PAGE, according to the method reported by Shagger and von Jagow(81) (Figure 7a). Following electrophoresis, the gels were either silver stained or transferred to nitrocellulose. Replicates were assayed with antisera directed to LHCII, CP29, CP26, and CP24(82) (Figure 7bd). Because of the high degree of homology shared by all the antenna polypeptides, it was essential for identication of the fractions to take into account both the immunoreactivity and the electrophoretic mobility of the SDS/PAGE bands. In TRIS/Tricine electrophoresis, antenna proteins migrate in the following order of increasing mobility: CP29 > CP26 > LHCII > CP24 and within LHCII, Lhcb1 > Lhcb2 > Lhcb3.(83) Accordingly, the slower-migrating band was detected by the anti-CP29 in peak 4 (Figure 7b), while peak 8, containing a slightly more mobile band, was recognized by anti-CP26 (Figure 7c). The anti-CP24 detected several bands (Figure 7d), but the strongest signal, with respect to the intensity of the band in the silver-stained gel, was obtained with the most mobile band in peak 3. Therefore, peaks 3, 4, and 8 seem to contain CP24, CP29, and CP26, respectively. The higher hydrophobicity of CP26 agrees with the longer elution time required. Thus, according to the migration order of increasing mobility reported above, the fastest migrating band was recognized as Lhcb3, the second fastest migrating band as Lhcb2, and the two newly resolved bands as Lhcb1 components of LHCII. From the above data, we conclude that peak 1 does not contain protein components; peak 2, the Lhcb2 component of LHCII; peak 3, the CP24 (Lhcb6); peak 4, CP29 (Lhcb4); peaks 5 and 6, two Lhcb1 components of LHCII; peak 7, Lhcb3; and peak 8, CP26 (Lhcb5). In order to support the identication previously assigned, the protein contained in each HPLC peak was subjected to amino acid microsequencing and compared with amino acid sequences reported in the literature for other species (data not shown). Good agreement with the above assignment was found. Finally, the assignment of each peak resolved by RPHPLC performed by electrophoresis, immunoblotting, and amino acid sequencing is corroborated from the values of molecular masses determined by the combined use of microbore HPLC coupled on-line with a mass spectrometer(84) equipped with an electrospray ion source (Section 6). From the results presented, it can be inferred that the major antenna system of PSII isolated from spinach leaves contains two different Lhcb1 proteins that can be resolved by the RP-HPLC system employing a 250-mm long Vydac C-4 column eluted with a multistep acetonitrile gradient and by the high-resolution TRIS/Tricine SDS/PAGE system in gels of 15 cm length (data not shown). On
18
MW MW (kDa) 66 45 36 29 24 20 14 (a)
(b)
(c)
(d)
Figure 7 Gel electrophoresis and immunoblotting of each protein collected throughout the chromatographic run reported in (a).
Lanes correspond to the peak labels in (a); the last lane (c) represents the LHCII from Zea mais used as a control. (a) SDS/PAGE of uniform polyacrylamide concentration (13%) with TRISTricine system of each collected peak, which were dried and then solubilized in 4% SDS, 120 mM DTT, and 120 mM TRIS-HCl (pH 8.45), containing 5 M urea. (b) Immunoblotting of the gel reported in (a) detected by using antisera directed to CP29. (c) Immunoblotting of the gel reported in (a) detected by using antisera directed to CP26. (d) Immunoblotting of the gel reported in (a) detected by using antisera directed to CP24. (Reprinted from Photosyntheses Research 61 (3), 1999, 281290, Zolla et al. Copyright (1999), with kind permission from Springer Science and Business Media.)
the other hand, the small microsequence performed does not reveal any amino acid differences, and the molecular mass measured by MS indicates that the difference is in the order of 6080 kDa. Further experiments and a complete amino acid sequence are necessary to explain these different subpopulations of type I. Nevertheless, the existence of more type I LHCII is in accordance with molecular genetic data reported in the literature showing that higher plants have several Lhcb1 genes encoding different Lhcb1 proteins for each species.(75) However, it is important to note that the resolution by SDS/PAGE of two Lhcb1 proteins usually requires special experimental conditions such as the use of polyclonal and monospecic antibodies,(85) dedicated electrolyte solutions, and a gel of extended length,(86) whereas using HPLC the two different subpopulations may be easily separated. The RP-HPLC method reported here, in addition to being rapid, simple, and precise, has proven to be effective at detecting differences in the protein components of LHCII isolated from different plants that might not be evidenced by denaturating SDS/PAGE, as in the case
of type I of spinach. This knowledge is expected to shed light on the composition and supramolecular organization of LHCII and may increase the understanding of the molecular mechanisms underlying the physiological adaptations of higher plants to environmental conditions.
5.2.6 Separation of Proteins Present in Photosystem I PSI prepared from the thermophilic cyanobacterium Synechococcus elongatus has been crystallized and the structure determined at a resolution of 6 A.(87) The determination of the structure of higher-plant PSI has been less successful, most likely because it is a much larger complex than its cyanobacterial counterpart. The cyanobacterial PSI complex consists of 11 subunits.(88) In the case of higher plants, although a lot of PSI genes have been identied and sequenced, there is little information about the actual proteins, and the relationship between gene and protein expression. This is related to the fact that the molecular masses of most of these proteins range
19
between 4 and 25 kDa, and consequently, they are not well separable by SDS/PAGE. 5.2.6.1 Isolation of Photosystem I by Sucrose Gradient Ultracentrifugation In the rst phase of experiments, supernatants were concentrated by ultraltration and then solubilized by -DM at a nal concentration of 1%. After stirring for 20 min at 4 C, the sample was centrifuged for 10 min at 40 000g, and 6-mL aliquots of the supernatant were loaded on 0.11 M sucrose gradients (35 mL) layered over 2 mL of 2 M sucrose, containing 5 mM Tricine (pH 7.8) and 0.03% -DM. After centrifugation for 42 h at 28 000 rpm in an SW28 rotor (Beckman) at 4 C, four green bands were distinguishable. The bands were collected and analyzed by SDS/PAGE.(89) The lowermost band, containing PSI-200, was diluted in 5 mM Tricine (pH 7.8) and centrifuged for 3 h at 70 000 rpm in an 80 Ti rotor (Beckman). The pellet was resuspended in 5 mM Tricine (pH 7.8) and 50 mM sorbitol, frozen in liquid nitrogen, and stored at 80 C. 5.2.6.2 Purication of Photosystem I Core and Lightharvesting Complex of Photosystem I The pellet from PSI-200 preparation was resuspended at 0.3 mg mL1 in distilled water and solubilized by 1% -DM and 0.5% Zwittergent-16. After stirring for 20 min at 4 C, the sample was rapidly frozen in liquid nitrogen and slowly thawed to improve the detachment between the PSI core and the light-harvesting complex of photosystem I (LHCI). Samples (1.5-mL aliquots) were loaded on a 12-mL 0.11 M sucrose gradient, also containing 5 mM Tricine (pH 7.8) and 0.03% -DM. After centrifugation for 24 h at 4 C at 41 000 rpm in an SW41 rotor (Beckman), ve green bands were obtained. The bands were analyzed by SDS/PAGE.(89) The second band from the top contained all the LHCI polypeptides, and the third band contained the PSI core particles. The fractions were frozen in liquid nitrogen and stored at 80 C. 5.2.6.3 Separation of Photosystem I Proteins by Reversedphase High-performance Liquid Chromatography The complete resolution of the protein components of the PSI was performed by RP-HPLC as for PSII. The experiments were carried out by performing chromatography of all proteins of PSI-200 and then separately for the antenna and core proteins, which had been previously isolated by sucrose gradient ultracentrifugation. Figure 8(ac) compares the chromatograms obtained upon injection of (a) isolated antenna proteins, (b) the core proteins, and (c) the total PSI-200.(89) Different gradients were used to nd the optimum experimental conditions to resolve the protein components of the antenna and core complex. In the case
Absorbance
45 40 35 30 25 20 15 10 5 0 20 40 Time (min) 40 35 30 25 20 15 10 5 0 20 40 Time (min) 120 60 80 60 80
(a)
(b)
Absorbance
Absorbance
100 80 60 40 20 0 PsaE PsaH.C 20 Lhca 1 3
Lhca4 Lhca2 PsaA+B PsaF PsaL,F,G
PsaD (c) 40 Time (min) 60 80
Figure 8 RP-HPLC separation of (a) spinach LHCI proteins contained in band 2 and (b) core proteins in band 3 of PSI-200 fractionated by sucrose gradient ultracentrifugation and (c) in whole PSI-200. Column, Vydac Protein C-4 (250 10 mm ID), eluted with an increasing acetonitrile concentration gradient as described in the text. Flow rate, 4.7 mL min1 ; detection at 214 nm. of PSI-200, the optimum separation of all proteins was obtained with a multistep gradient. Good complementarity between the three chromatograms is observed, indicating that the presence of all proteins in the PSI200 system does not affect the elution time of each protein. The material harvested from the second band of sucrose gradient ultracentrifugation is resolved into ve main peaks (Figure 8a). Previous analyses with SDS/PAGE showed that it contained a mixture of the protein components of the PSI antenna system. Most of the chromatographic peaks are symmetrical, but the last one at 43.4 min is broad, indicating the presence of more than one protein. In contrast, the material harvested from the third band of the sucrose gradient ultracentrifugation, containing essentially the core protein components of
20
PSI, shows a main peak at a long elution time of 61 min and numerous smaller peaks. The main peak is broad and elutes at a long time, suggesting the presence of more than one protein showing high hydrophobicity. Finally, in the chromatogram for the total PSI-200, ve of the same peaks are present, with retention times corresponding to those observed in Figure 8(a), which are the most abundant separated proteins and allow one to hypothesize that they represent the antenna complex, although only four proteins are expected. Moreover, the main peak observed in the core complex (Figure 8b) is also observed in Figure 8(c), which may be hypothesized to contain psaA and psaB, and other smaller core peaks are underestimated when all proteins are present. Once the optimized conditions to obtain a better separation of each component had been found, we attempted the identication of each protein contained in each peak by coupling on-line HPLC with MS (Section 5). The mass values measured by ESI-MS corresponded to the molecular mass expected on the basis of the DNA sequence, allowing the assignment of each peak as reported in Figure 8(c).(89) These results corroborate the utility of coupling an HPLC/MS on-line with an electrospray ion source. Finally, the separation of both PSI and PSII may be achieved by direct injection of thylakoid membrane into the column and elution with a multistep gradient. All the protein components of PSII can be well resolved without interference from the other protein components of PSI, indicating that the use of the crude thylakoid membrane preparation does not affect the resolution and retention times of each protein. 5.2.7 Comparison of Monolithic and Packed Capillary Columns in the Separation of Photosystem I Components An analytical study of the loading capacity of the monolithic stationary phase with that of a beaded, totally porous, PS/DVB stationary phase (Vydac 259VHM, 5 m, 300 A) revealed that the loading capacities of the monolithic and the beaded stationary phases are about the same for large proteins of Mr > 50 000, whereas the amount of polypeptide of Mr < 15 000 that can be loaded onto the porous stationary phase is approximately 10-fold that of the monolithic one.(90) The reason for this discrepancy is the difference in accessibility of the chromatographic surface to analytes of different molecular size. The surface in the micropores of porous separation media is not accessible to large biomolecules; thus, the loading capacity decreases rapidly with increasing molecular mass. Moreover, compared to octyl or octadecyl stationary phases, PSDVB monolithic stationary phases are known to be mildly
hydrophobic(91,92) , which makes them eminently suitable for the separation of very hydrophobic membrane proteins. In this review,(93) the applicability of the RPHPLC/ESI-MS method (using both monolithic capillary and beaded columns) to the analysis of larger proteins in biological samples in a difcult matrix is demonstrated using the protein components of PSI of thylakoid membranes whose molecular masses range between 3500 and 80 000. Figure 9 compares the separation of all PSI components performed by capillary monolithic and a beaded silica capillary lled with C4 RP. The separation time of the monolithic column is almost halved compared to that expected of the same protein mixture on a conventional, butyl-silica stationary phase with 300-A pores, and the resolution of the peaks is signicantly better. The peak widths at half-height ranged from 3 to 5 s, which demonstrates the high resolving power and speed of analysis with monolithic PS/DVB capillary columns. However, in both cases, mass spectra of high quality were extracted from the reconstructed ion chromatograms, showing no adduction with detergent. Interestingly, the elution order changed from Lhca1< Lhca3< Lhca4< Lhca2< PsaF on the silica-based stationary phase to Lhca1< Lhca4< Lhca3< PsaF< Lhca2 on the monolithic column, which points to differences in selectivity between the silica-bonded and the polymeric stationary phase. 5.2.8 Reversed-phase High-performance Liquid Chromatography/Electrospray Ionization Mass Spectrometry of Entire Thylakoid Membranes In this last section, we present the results of a study designed to develop a rapid and straightforward method, using HPLC, to resolve and identify the protein components of both the PSI and PSII, starting from the entire thylakoid membrane. Normally, before loading a complex biological matrix on any HPLC system, membrane proteins must be preseparated according to their hydrophobic characteristics, and this is achieved by selective extractions. This pretreatment facilitates subsequent separation and provides a rst guideline for the choice of detergent. As a rule, the detergent chosen for the running buffers is the same one with which the protein was solubilized. Also, in order to retain the biological activity of proteins, it is preferable to use less denaturing detergents and nonionic detergents for solubilization and in the running buffers (the interested reader is referred to the article by Seddon et al.(8) for further details on the use of detergents for membrane protein analysis). The presence of detergent in all steps minimizes the tendency toward association and aggregation as well as the possibility of nonspecifc interactions with the support used for chromatographic separation.

Lhca4 Lhca2.1 Lhca2.2
21
PsaA: PsaB PsaL
Absorbance at 214 nm
120
PsaC; PsaE; PsaN Lhca3 PsaG
PsaH
PsaD
Lhca1.1 Lhca1.2
80
PsaK
40
0 0 1.2
psa N
(a)
10
20
30
40
PsaF
50
60
Absorbance at 214 nm
psa D
lhca 3 psa F lhca 2.1 lhca 2.2
psa H psa C psa E
lhca 1.1 lhca 1.2
psa G
lhca 4
0 (b) 0 10 20 Time (min) 30 40
Figure 9 Comparison of RP analysis of PSI subunits from barley performed by PS-DVB-monolith and C4-beaded column.
(a) Column, PS-DVB-monolith, 60 0.10 mm ID; mobile phase, (solvent phase A) 0.050% triuoroacetic acid in water, (solvent phase B) 0.050% triuoroacetic acid in acetonitrile; linear gradient, 3949% B in 20 min, 4655% B in 10 min, and 5599% B in 15 min; ow rate, 2.0 L min1 ; and temperature, 60 C. (b) C4-beaded capillary, 150 0.18 ID; mobile phase and gradient as (a); ow rate, 2.0 L min1 ; and temperature, 25 C.
The rst consideration in dening an HPLC method development strategy is to establish if we need proteins with their biological properties intact or if we simply need to characterize them. There are many factors to be taken into consideration during the separation development process in order to select appropriate conditions and an appropriate detergent, under which artifactual denaturation is minimized. Factors arising from the presence of detergent must be taken into account along with the possibility of nonspecif` interactions c with the support used for chromatographic separation. Because of the high complexity of the photosynthetic apparatus, liquid extraction followed by sucrose gradient ultracentrifugation was chosen as the sample preparation and prefractionation method.(93) The membrane proteins held in the suspended thylakoid membranes at a nal concentration of 1 mg chlorophyll per mililiter were solubilized by adding DM (dodecil maltoside) to a nal concentration of 1% (w/v). After stirring for 20 min at 4 C, the sample was centrifuged for 10 min at 20 000g to eliminate insoluble material. One-mililiter aliquots of the supernatant were loaded onto 12-mL ultracentrifugation tubes containing 0.5 mol L1 sucrose, 5 mmol L1 Tricine,
psa K
pH 7.8, and 0.030% DM. After centrifugation (rotor TST 41.14) for 42 h at 140592 g at 4 C, four green bands were distinguishable. The green bands were harvested with a syringe. SDSPAGE analysis of the fractions from spinach revealed that fraction 1 contained a mixture of the protein components of the minor and monomeric major PSII antenna systems, whereas fraction 2 essentially contained the protein components of the trimeric major PSII antenna system, as previously reported. Fraction 3 contained the reaction-center complex of PSII, and nally, fraction 4 contained the subunits of PSI.(93) The bands were subsequently analyzed by RP-HPLC/ESI-MS using PSDVB-based monolithic capillary columns. Monolithic capillary columns (60 0.20 mm ID and 60 0.10 mm ID) were used for protein separation. ESI-MS was performed on a quadrupole ion trap mass equipped with a triaxial electrospray ion source. An electrospray voltage of 1.62.0 kV and a nitrogen sheath gas ow were used for analysis with pneumatically assisted ESI. Fine tuning for ESI-MS of proteins was accomplished by infusion of a 6.9 pmol L1 solution of carbonic anhydrase in 0.050% aqueous TFA solution containing 20% acetonitrile (v/v) at a ow rate of 3.0 L min1 .
psa L
psa A/B
22
The analytical method was shown to be very exible and allowed the antenna proteins to be identied, as well as most of the proteins of the reaction center from PSI and PSII in various plant species, requiring few RPHPLC/ESI-MS analyses and only minor adaptations in the acetonitrile gradients in 0.05% aqueous TFA. The membrane proteins, ranging in molecular mass from 4196 (I protein) to more than 80 000 (PSI A/B), as well as isoforms, were identied on the basis of their intact molecular masses and comparison with molecular masses deduced from known DNA or protein sequences. Figure 10 depicts the RP-HPLC/ESI-MS analysis of PSII reaction-center subunits contained in fraction 3. Chromatography at elevated temperatures facilitated
1.4
65 C
(a)
0 1.9 74 C
(b)
0 1.7 78 C
0 0 (c) 10 20 Time (min) 30 40
Figure 10 RP-HPLC/ESI-MS analysis of PSII core proteins of spinach performed at different temperatures. Column, PS-DVB-monolith, 60 0.20 mm ID; mobile phase, (solvent phase A) 0.050% triuoroacetic acid in water, (solvent phase B) 0.050% triuoroacetic acid in acetonitrile; linear gradient, 050% B in 30 min and 5062% B in 10 min; ow rate, 2.5 L min1 ; temperature, (a) 65 C, (b) 74 C, and (c) 78 C; detection, ESI-MS, scan, and 6002000 amu; electrospray voltage, 1.6 kV; sheath gas, nitrogen; and sample, fraction 3 of spinach.
elution of the membrane proteins as very sharp peaks, although a relatively shallow gradient was applied for elution. Most of the proteins of the reaction center were well separated at 65 C but the large proteins psb C and psb D co-eluted at this temperature (Figure 10a). Nevertheless, a shoulder appeared in the peak eluting around 35 min at 74 C, and the proteins were almost separated to baseline at 78 C (Figure 10c). The molecular masses of the subunits obtained upon deconvolution of the raw mass spectra that were extracted from the reconstructed total ion current (TIC) chromatogram were in excellent correspondence with the molecular masses expected from the DNA sequence (including posttranslational modications), wherever relevant. Thus, high- and low-molecular-mass PSII reaction-center subunits were successfully analyzed by ESI-MS following off-line RPHPLC fractionation.(94) Nevertheless, molecular mass determination was relatively inaccurate because of coeluting compounds, especially in the high mass range.(95) Moreover, some of the high-molecular-mass subunits, such as psb C or psb D, could not be detected at all.(95) Because of the high hydrophobicity of the psb A and psb D subunits, they were difcult to elute from an octyl-silica column (Aquapore RP-300) with 1propanol/5% acetic acid.(95) Because of the high chemical stability of the polymeric stationary phase, analysis with PS-DVB monoliths can be performed routinely at elevated temperatures, which signicantly enhances chromatographic performance and elution characteristics of proteins. High-quality mass spectra made it possible to identify and quantify the nonphosphorylated and phosphorylated reaction-center subunits D1, D2, and CP43 of PSII, containing ve to seven membrane-spanning -helix. A semiquantitative estimation of protein phosphorylation was made by comparison of the relative signal intensities of the phosphorylated and nonphosphorylated protein mass peaks. psb A and psb D showed quite similar percentages of phosphorylation, namely, 42% and 39%, respectively, whereas psb C was predominantly present in the phosphorylated form (69%).(95) The measured molecular mass of psb H (7698) indicated the presence of one phosphorylation and one oxidation. Thus, because of its great exibility and suitability for proteins having a very wide range of molecular masses and hydrophobicities, the method is generally applicable to the analysis of complex mixtures of membrane proteins. Moreover, intact mass measurements by ESIMS provide mass accuracy, often exceeding 0.01% (100 ppm), and resolution sufcient to observe the rst oxidation adduct (16 Da) or phosphorylation (79 Da), providing an attractive alternative technique to monitor the subtle changes that often accompany physiological
Signal intensity (counts 108)
Detergent
psb O
psb F psb R
psb E psb L
psb B psb H psb I psb K psb A psb C psb D
23
adaptations. This presents a new facet to intact mass proteomics as a potent tool in proteomics. 5.3 Separation of Lipoproteins 5.3.1 Preparation of Lipoprotein Fractions Plasma or serum can be used for lipoprotein analysis. However, plasma is preferred since it can be kept cold in order to slow most enzymatic processes that degrade lipoproteins. The following procedure is commonly used to prepare lipoproteins.(96) Human blood (Ca 2030 mL) was collected from normolipidemic males by venipuncture after 1216 h of fasting. The blood was allowed to stand for 23 h at room temperature until agglutination was complete. Plasma was withdrawn after centrifugation at 1000g at 15 C for 15 min. The plasma density was adjusted to 1.225 g mL1 by adding solid potassium bromide (0.3517 g KBr per milliliter of plasma). Plasma (1.225 g mL1 , Ca 35 mL) was then placed in ultracentrifuge tubes, which were centrifuged in a swinging-bucket rotor at 200 000g at 10 C for 40 h. The lipoprotein fraction prepared by this procedure did not contain serum proteins, except for a small amount of albumin. The lipoprotein fraction in the KBr solution was dialyzed against 0.154 M sodium chloride solution. 5.3.2 Preparation of Individual High-density Lipoprotein, Low-density Lipoprotein, and Very Low-density Lipoprotein Fractions Many separations subfractionate the lipoprotein fraction of plasma. Hence, each main class of lipoproteins may be collected by ultracentrifugation using a multiple discontinuous density gradient. For this purpose, human blood was collected from fasting normolipidemic healthy males in tubes containing 0.15% EDTA. The plasma was separated by centrifugation at 700 rpm at 7 C for 20 min. A discontinuous NaClKBr density gradient (total volume 18.5 mL) was formed by adjusting the density of plasma to 1.30 g mL1 with solid KBr and sequentially layering on the adjusted plasma salt (NaClKBr) solutions with densities of 1.240, 1.063, 1.019, and 1.006 g mL1 and 0.5 mL of distilled water. Tubes loaded with the discontinuous density gradient were placed in a vertical rotor and centrifuged at 313 500g at 7 C for 80 min. 5.3.3 Separation of Particles and Subfractions In an alternative to ultracentrifuge separation, the GPC mode of HPLC is the most commonly used method to separate particles such as HDL, VHDL, and LDL. Two commercial columns are commonly used: Superose 6 (from Pharmacia) and TSK type. Rigid, fast-ow agarose
gels, such as Superose 6, are able to accept large sample volumes (up to 5 mL) and preferred for the preparation of larger quantities of plasma lipoproteins. Furthermore, multiple automated separations of lipoproteins from whole plasma may be performed with a preparative-grade Superose 6 column. Thus, 2 mL of plasma was loaded and eluted with 0.9% sodium chloride and 2 mM sodium phosphate (pH 7.4). The eluent also contains 0.02% or 1% sodium azide for serum or plasma separation, respectively. VLDL, LDL, and HDL can be separated in a single run. A single separation can be completed in about 160 min. Hence, the system can be used to analyze up to six different plasma samples (2 mL per sample) overnight. Fast-ow separation of plasma is also used, where lipoproteins are detected on-line at 500 nm after postcolumn derivatization with an enzymatic cholesterol reagent (CHOD-PAP, Boehringer Mannheim). The eluting lipoproteins and the cholesterol reagent are mixed in a chamber attached to the column outlet. The mixture is then passed through a reaction capillary and detected. Complete separation of lipoprotein fractions is usually achieved in<80 min. The chromatographic proles of normal and several types of hyperlipoproteinemic (HLP) serum samples are illustrated in Figure 11(ad). The three peaks correspond to, in order, VLDL, LDL, and HDL.(97) Separation of lipoproteins by HPLC on TSK-type gel permeation columns is faster than on a Superose 6 column, but smaller sample volumes (<300 L) may be loaded. Furthermore, serum instead of plasma is preferred for TSK-gel HPLC of lipoproteins. With plasma, there is a danger of brin forming during the analysis. A further disadvantage of the TSK method is that this type of column is generally more prone to clogging than a Superose 6 column. Adsorption of lipoproteins on the support has also been noted by some workers. It was found that a combination of G4000 SW and G3000 SW eluted with 0.15 M NaCl (pH 7) was best at separating serum lipoproteins into VLDL, LDL, HDL2 , and HDL3 .(98) Separation by a TSK column allows the determination of lipids using a combination of two TSK columns and on-line enzymatic reaction. The system used two detectors. The rst was placed immediately after the column and monitored protein absorbance at 280 nm. The second detector was placed after the enzymatic reactor and detected lipid absorbance at 500600 nm.(98) Separation of large amounts of LDLs and VLDLs from human serum may be performed by using a hydroxyapatite Bio-Gel HTP DNA grade column (25 1.0 cm ID) by one or four stepwise elutions.(36) In the latter case, a 2-mL volume of human serum is loaded on the column and eluted with a discontinuous gradient of 75, 200, 300, and 650 mM potassium phosphate buffer
24
500 400 mV mV 300 200 100 0 0 10 20 30 40 50 60 70 80 (a) 700 500 mV mV 300 100 0 10 20 30 40 50 60 70 80 (c) Retention time (min) (d) 0 10 20 30 40 50 60 70 80 Retention time (min) 300 200 100 Retention time (min) (b) 0 10 20 30 40 50 60 70 80 Retention time (min) 500 400 300 200 100
Figure 11 Chromatographic prole of normal and HLP sera. Conditions: 300-mm Superose 6 column eluted with 100 mM Na2 HPO4 and 200 mM NaCl (pH 7.4) at 300 mL min1 . (a) Normolipidemic sample; (b) type IIa HLP; (c) type III HLP; and (d) type IV HLP. Peaks from left to right: VLDL, LDL, and HDL. (Reproduced by permission of Clinical Chemistry from W. Marz, R. Skeimer, H. Scharnagi, U.B. Seiffert, W. Gross, Clin. Chem., 39(11), 22762281 (1993).)
650 mM 700 Concentration of KPi (mM) 2.0 Absorbance (280, 580 nm) 75 mM KPi (pH 7.4) HDL serum products 1.5 400 1.0 Serum 2.0 mL inject 0.5 580 nm 200 nm 0 0 20 40 60 80 Elution volume (mL) 100 120 0 Serum products LDL VLDL 100 300 200 200 mM 300 mM 500 600
Figure 12 Stepwise elution prole of human serum lipoproteins. Column, Bio-Gel HTP DNA grade 25 1.0 cm ID; eluents, 25, 200, 300, and 650 mM potassium phosphate buffer (KPi) (pH 7.4); owrate, 12.0 mL h1 ; and sample, 2.0 mL of human serum. (Reproduced by permission of Elsevier Science from Y. Shibusawa, J. Chromatogr. B, 699, 419437 (1997).) at pH 7.4. Figure 12 shows the elution prole of human serum obtained with a hydroxyapatite column. Four peaks are detected, the rst containing HDLs and serum proteins; the second, the serum proteins, eluted at 200 mM; the third, mainly LDLs, eluted at 300 mM; and the fourth, VLDLs, eluted at 650 mM. The whole lipoprotein fraction (d < 1.21) can be further subfractionated into VLDL, LDL, and HDL by GPC.(40) Lipoprotein fractions prepared by ultracentrifugation are often used for this further chromatographic separation. In this way, the procedure is both analytical and preparative since the isolated fractions contain no contamination from serum proteins. Subfractionation of HDL particles accomplished by GPC gives rise to subfractions having different sizes (3.81528 nm) and different amounts of proteins: HDL2a , HDL2b , HDL3b , and HDL3c .(40)
25
HDL particles may also be subfractionated by heparin afnity chromatography according to the apoE content.(42) HDLs rich in apoE were separated into ve subfractions using an elution buffer with varying Mn2+ concentration.(41) In the case of LDL, charge heterogeneity was demonstrated by ion-exchange chromatography. These types of separations are important since oxidatively modied LDLs have been associated with atherosclerosis.(91) LDL particles modied by Cu2+ were analyzed by anion-exchange chromatography using a Mono Q-HR 515 column (Pharmacia).(43) Five different oxidatively modied LDLs were identied. The isolated fractions exhibited differences in density and lipid content. 5.3.4 Separation of Apolipoproteins and Isomeric Forms The growing evidence that apolipoproteins are better markers of CHD than serum cholesterol levels(35) has increased the interest in analyzing lipoproteins according to the apolipoprotein compositions. Afnity chromatography, which employs specic and reversible interactions between lipoproteins and ligands, is capable of separating lipoproteins on the basis of their apolipoprotein content by using nonspecic binding and IA. Heparin was successfully immobilized on to glyceryl CPG and used to bind specically -lipoproteins (apoB).(98) Serum samples applied to heparinCPG were separated into two fractions. The unretained fraction was washed through the column with 0.1 M NaCl, and the retained fraction was eluted with 1 M NaCl. Radial immunodiffusion studies conrmed the complete resolution of apoA- and apoB-containing lipoproteins. However, attempts at incorporating the heparinCPG column into a high-performance afnity chromatography system were unsuccessful. Two types of lipoproteins have been clearly established as atherogenic, the apoB100containing LDL and apoB48-containing chylomicron remnants.(99) Human genetic disorders resulting in increased circulating levels of either of these lipoproteins cause premature atherosclerosis. Experiments were reported to demonstrate that the NH2 -terminal region of apoB binds to heparin afnity gels with an afnity equal to or greater than that of apoB100-containing LDL.(100) In addition, apoB48-containing lipoproteins were observed to bind to heparin and LDL. Hence, based on these ndings, it was proposed that NH2 -terminal apoB contributes to the atherogenicity of LDL and remnant lipoproteins. Dextran sulfate is a synthetic analog of heparin, which binds preferentially to apoB-containing lipoproteins. Commercial sulfated dextran beads from Sigma consist of sulfated cross-linked dextran and are useful for
rapid (15 min) separation of apoA- and apoB-containing lipoproteins on elution of proteins by NaCl at 80 mM. IA chromatography provides the highest specicity for separation and allows the separation of relatively large volumes (110 mL) of whole plasma or lipoproteins. Preparations of both polyclonal antibodies(101) and MAbs(102) to apolipoproteins, produced using puried delipidized apolipoproteins, are commonly used as antigens. Antibodies to apolipoproteins coupled to cross-linked dextran (Sephadex) or agarose derivatives (Sepharose) serve as selective ligands for the separation of lipoproteins by IA chromatography. Separation of apoproteins into their isomers is of interest especially for apoA and apoB, which may be performed by sequential chromatographic stages using more IA or afnity chromatography followed by gel permeation, ion-exchange, or RP techniques. In the rst procedure, whole plasma is rst fractionated into apoB and apoA lipoproteins by afnity chromatography on concanavalin A (con A). Further subfractionation proceeds by subsequent IA chromatography using a specic antibody: anti-apoA-II immunosorbent, which retains a fraction containing lipoprotein (A-I + A-II) and lipoprotein A-II. Further, separation of this fraction on an anti-apoA-I immunosorber leads to the isolation of three types of apoA lipoproteins: lipoprotein A-I, lipoprotein A-II, and lipoprotein (A-I + A-II).(103) Conventional GPC or ion-exchange chromatography on soft gel supports can be used for the preparation of large quantities (1050 mg) of pure apolipoproteins,(104) whereas by HPLC, only small amounts of sample (<5 mg) can be applied to the column but the analysis is completed within 60 min. Separation by high-performance GPC is strongly dependent on the completeness of the delipidation step, which may be performed satisfactorily with an organic solvent. However, high-performance GPC separation of HDL apolipoproteins has been achieved without prior organic solvent delipidation of the HDL fraction.(105) GPC is often used as a preparative technique for isolating apolipoproteins derived from HDL or VLDL, while HDL apolipoproteins, apoA-I, apoA-II, and apoC, can be separated on Sephadex G-200 columns using TRIS-HCI buffer (pH 8.6) containing 8 M urea.(106) apoC and apoE from VLDL can be isolated by Sepharyl S-200 (Pharmacia) chromatography.(106) Separation of apolipoprotein isoforms can be achieved by either ionexchange or RP-HPLC. Apoc isoforms (C-III0 , C-III1 , and C-III2 ) can be separated on Mono Q-HR 5/5 (Pharmacia) anionexchange columns(107) in less than 30 min using a linear gradient of 0.15 M NaCl in TRIS-HCl containing 6 M urea (pH 8.2). This mode of apolipoprotein separation is based on differences in isoelectric points. Chromatography
26
Absorbance at 215 nm (mAU) 1000
500 A-IV
0 0 5 10
C's A-I 15 Time (min) 20
E 25
Large throughpores allow convective ow through the particles, quickly carrying sample molecules to short diffusive pores inside. By reducing the distance and therefore the time over which molecules diffuse to access the particle-binding surface area, the ow rate can be increased 10- to 100-fold with little or no loss in resolution or capacity. Thus, once a particular mode of chromatography has been selected, it is possible to perform it by using perfusion particles. In general, the perfusion chromatography allows routine achievement of high-resolution laboratoryscale separations in 30 s3 min; rapid and systematic determination of the best separation method for optimum purity; a signicant reduction in the time to process largevolume samples; improved yields and recoveries of biological activity through faster processing. 6.1.2 High-performance Liquid Chromatography Coupled On-line with Mass Spectrometry Further developments in HPLC system have been achieved by coupling the outlet on-line with other instrumentation. The introduction of electrospray ion sources that are coupled with quadrupole mass lters has produced a mass spectrometer that is easily compatible with HPLC and CE, and therefore, analysis and measurement of biological samples at a reasonable cost may be performed.(113,114) Since most MS ion sources require lower ow rates than common chromatographic levels, a split of the HPLC eluent stream must be accomplished. Such systems are highly advantageous in that they facilitate the simultaneous separation of complex mixtures along with ultraviolet (UV) adsorption and mass detection. With regard to the separation of thylakoid proteins of the PSII, RP-HPLC interfaced with MS with an electrospray ion source allows the separation and accurate molecular mass determination of the individual membrane proteins contained in each peak on the HPLC trace, in particular, for the identication of the major LHCII and minor (CP24, CP26, and CP29) antenna system, whose molecular masses range between 22 and 29 kDa.(85) Table 2 reports the comparison of the molecular mass values calculated from the protein sequence derived from the isolated genes with those determined by RPHPLC/ESI-MS together with the apparent molecular masses given by SDS/PAGE. The molecular mass values determined are in good agreement with the computed molecular masses of these proteins based on their DNA sequences.
Figure 13 RP-HPLC separation of delipidated rat HDL (200 mg protein) on a TSK Phenyl-5PW column. Gradient between 20 mM H3 PO4 in water (pH 2.3) and 20 mM H3 PO4 in 60% acetonitrile. Flow rate, 1 mL min1 at 45 C. Fractions were analyzed by SDS/PAGE and their lipoprotein contents as assessed by Coomassie brilliant blue staining (bold bars) and by silver staining (normal) are as indicated. (Reproduced by permission of Elsevier Science from B. Meyer, E. Kecorius, P. Barter, N. Fidge, T. Tetaz, J. Chromatogr. A, 540, 386391 (1991).) of VLDL and HDL apolipoproteins is performed on anion-exchange columns such as SynChropak AX300 (SynChrom IUC) and Mono Q-HR (Pharmacia).(108) A rapid and highly efcient technique for monitoring heterogeneity in apolipoproteins is the separation on the basis of differences in hydrophobicity by RP-HPLC. Unfortunately, the small amounts of apolipoprotein (50100 g), which may be loaded, render this method qualitative but highly resolutive. Successful separations of apoA and apoC were performed on C-18 columns with a gradient of acetonitrilewater in the presence of 0.1% of TFA.(109) Figure 13 shows the separation of human apolipoproteins A-IV, A-I, and E on a TSK Phenyl-5PW column.(110)
CURRENT TRENDS
6.1 Membrane Proteins Most of the methods described in previous sections, although seemingly outdated, still represent valid, if not unique, approaches to delve into the complexity of membrane proteins.(111) 6.1.1 Perfusion Chromatography The advent of perfusion chromatography, which consists of matrix containing ow-through particles, has further increased the interest in using HPLC systems, especially for membrane proteins.(112) In contrast to conventional particles, the new matrix has two classes of pores.
27
Table 2 Comparison between experimental and computed values of the molecular masses (Da) of the protein constituents of the PSII major and minor antenna system
Antenna proteins Type 1 I Type 1 II Type 2 Type 3 CP 29 CP 26 CP 24
a
SDS/PAGE: apparent molecular mass (Da) 27 00028 000 25 00027 000 24 00025 000 29 00031 000 26 00029 000 20 00022 000
Molecular mass (measured) 24 936 24 942 24 761 24 323 28 076 27 068 22 820
Molecular mass (expected) Spinach 25 030 22 813 Other speciesa 25 026 (T) 24 969 (M) 25 041 (T) 24 906 (P) 24 834 (T) 24 285 (B) 24 308 (T) 27 804 (B) 27 642 (T)? 26 607 (M) 27 642 (T)?
Plant species: P, petunia; M, maize; T, tomato; B, barley.
Thus, the assignment of each peak resolved by RP-HPLC performed as above by electrophoresis, immunoblotting, and amino acid sequencing is corroborated by the values of molecular masses determined by the combined use of HPLC coupled on-line with a mass spectrometer equipped with an electrospray ion source (ESIMS). Furthermore, the resolution of two variants of type I proteins is in agreement with previous ndings reporting the resolution of more than one type I protein by both high-resolution polyacrylamide gel electrophoresis(81) and HPLC(80) and is consistent with the high copy numbers of Lhcb1 genes isolated in higher plants,(75) giving condence in the real potential of these coupled techniques. On the other hand, the isolation of multiple copy numbers of the same gene from several higher plant species accounts for the resolution of different variants of the same proteins reported in the literature.(75) In the case of glycoproteins, ESI-MS can detect whether an oligosaccharide is O- or N-linked.(115) It can also differentiate between complex, high-mannose, and hybrid forms. Moreover, this technique may be used to gain limited linkage order information using collision-induced dissociation (CID) with both a single- and a triplequadrupole mass spectrometer. In fact, in a complex map, the region of the map that contains the glycopeptides can be deduced by looking for characteristic patterns in the 2-D plot or by the observation of oxonium ions produced by CID. The plot of m/z against retention time (contour maps) as a facile approach to the rapid 2-D mapping of complex samples has some similarity to the popular 2-D techniques currently used in biochemistry, such as a combination of IEF and SDS/PAGE, but offers a different combination of orthogonal separation methods. Such maps are readily available from the data generated by an HPLC/MS analysis and can give valuable information about glycosylation patterns and product consistency.
This new analytical method can be used to deduce possible carbohydrate structures by determining both the mass and elution position of individual glycopeptides. RP-HPLC/ESI-MS has greatly expanded the power of peptide mapping to identify carbohydrate structures that are attached to asparagine, serine, or threonine residues. One can use in-source CID to scan the map for regions with a high concentration of glycopeptides. Such information should prove invaluable in determining the role in the biology of the carbohydrate moiety in glycoproteins and in reducing the approval barriers for the pharmaceutical use of glycosylated proteins produced by mammalian fermentation systems. 6.1.3 High-performance Liquid Chromatography Coupled On-line with Light Scattering, Ultraviolet Absorbance, and Refractive Index Detectors Since protein molecules self-associate to oligomers for specic purposes and we often have no idea whether the protein exists in solution as a monomer, dimer, or other oligomer, the possibility of determining the molecular weights of proteins or their complexes is an important step in understanding proteins and their functions. In addition to HPLC coupled on-line with a mass spectrometer, techniques of using SEC with on-line light-scattering, UV absorbance, and refractive index detectors have recently been reported.(115) Conventional SEC is a simple and fast method for estimating the molecular mass of a protein in its native form based on its elution position. However, there are several problems with this simple SEC approach. One is that the elution position depends not only on the molecular mass of the protein but also on its shape. Another problem is that the elution position will change if the protein has any tendency to interact with the column matrix. In addition, when a
28
protein or a protein complex contains carbohydrates, the carbohydrates usually have a disproportionately large effect on its elution position, so SEC may not be able to determine its polypeptide molecular mass. However, as reported by Wen et al.,(116) by using two or more detectors, proteins and glycoproteins may be easily determined. 6.1.4 Afnity Chromatography and Biosensors Afnity chromatography, which in the past yielded success as a preparative tool, has stimulated the development of analytical applications for biomolecular recognition and most recently of molecular recognition biosensors. Afnity-based methods on other solid-phase surfaces such as blots and microliter plates have further expanded afnity-based technology. The principles of analytical afnity chromatography have recently been adapted for a nonchromatographic afnity technology, namely, real-time optical biosensors for interaction analysis. As a real-time method, biosensors offer the opportunity to measure not only the equilibrium afnity constant but also the on and off rate constants for interactions of biological macromolecules.(117) 6.1.5 High-performance Liquid Chromatography/Mass Spectrometry for protein quantitation The nal step of the classic experimental proteomics workow is characterized by protein/peptide identication (and/or sequencing) throughMS. For in-depth description of MS techniques, the interested reader is referred to other chapters of this encyclopedia. In this context, it will sufce to say that a mass spectrometer roughly includes an ionization source, a mass analyzer, and a detector. The ionization source produces gaseous ions from molecules in either a solution or a solid phase. Two of the most common sources are electrospray ionization (ESI) and MALDI (matrix-assisted laser desorption/ionization). The mass analyzer measures the mass-to-charge ratio of these ionized molecules. The most common mass analyzer (time-of-ight, TOF) determines the mass-to-charge ratio by measuring the time required for the ions to pass through a charged eld. While application of MS to identication of proteins/ peptides is long known, recent trends in proteomics target the quantitative rather than (or in addition to) the qualitative issue. Yet it still is an almost unexplored territory, which is of growing pivotal interest especially in the quest for new biomarkers of pathological conditions. A detailed review about the emerging role of quantitative techniques has been recently published.(118) Recent applications of MS techniques allow quantitation of protein species in the sample under analysis,
such as cysteine labeling by isotope-coded afnity tagging (ICAT) and isotope tagging for relative and absolute quantitation (iTRAQ). ICAT relies on chemical labeling reagents (ICAT reagents) consisting of three general elements: a reactive group that labels a dened amino acid side chain (e.g. iodoacetamide to modify cysteine residues), an isotopically coded linker, and a tag (e.g. biotin) for the afnity isolation of labeled proteins/peptides. For the quantitative comparison of two proteomes, one sample is labeled with the isotopically light probe and the other with the isotopically heavy version. To minimize error, both samples are then combined, digested with a protease (i.e. trypsin), and subjected to avidin afnity chromatography to isolate peptides labeled with ICAT reagents. These peptides are then analyzed by liquid chromatography/MS. The ratios of signal intensities of differentially mass-tagged peptide pairs are quantied to determine the relative levels of proteins in the two samples. iTRAQ is based on the covalent labeling of the N-terminus and side chain amines of peptides from protein digestions with tags of varying mass. The samples are then pooled and usually fractionated by nano-liquid-chromatography and analyzed by tandem MS. A database search is then performed using the fragmentation data to identify the labeled peptides and hence the corresponding proteins. The fragmentation of the attached tag generates a low-molecular-mass reporter ion that can be used to relatively quantify the peptides and the proteins from which they originated. It is relevant to underline that both the ICAT and iTRAQ approaches rely on intermediate HPLC fractionation methods, involving ion-exchange chromatography and MudPIT-like strategies (2-D-RP-SCX-HPLC). This is relevant in the light of the renewed interest growing around chromatographic approaches, as long as new and pivotal analytic strategies are introduced. Although quantitative results could also be obtained with gel-based approaches, as, for example, changes in quantities of platelet proteins during storage with uorophore-labeled 2-D-DIGE (differential in-gel electrophoresis), the obtained results are not as much as accurate. Indeed, one approach appears to be more suited to investigate proteins, while the other is more sensitive to changes in peptide concentrations, making it necessary to adopt an integrated strategy (or to choose the eligible one) depending on the fraction of interest. 6.1.6 Posttranslational Modications Other than quantitative data, modern researchers are growingly interested in the study of posttranslational modications in order to gain functional and physiological insights through protein-targeting studies. Indeed, as many as 300 posttranslational modications of proteins
29
are known to occur physiologically. Eukaryotic plasma membrane proteins are often heavily posttranslationally modied in the extracellular domains (e.g. Nglycosylation) and in the intracellular domains (e.g. phosphorylation or acylation). MS is a central technology in the protein chemists toolkit, enabling site mapping and quantication of chemical modications on proteins, as well as detection of new types of structures. Key to analyzing posttranslational modications by MS is to understand their chemical behavior in solution and gas-phase reactivities, given that the range in chemical behavior of amino acids and functional groups causes signicant differences among peptides with variable composition. An interesting and updated review of recent strategies toward posttranslational modications, in particular, glycosylations (see Sections 2.2.3 and 6.1.2), phosphorylations, acetylations, and ubiquitinations, has been proposed by Witze et al.(119) Although it seems to be distantly related to chromatography, albeit tied to MS, the study of posttranslational modications often relies on the preliminary enrichment of species of interest, such as of phosphorylated peptides or proteins. Low sensitivity is a frequent obstacle when analyzing phosphopeptides or phosphoproteins by MS. Substoichiometric phosphorylation often occurs, reducing phosphoanalyte abundances compared to corresponding unphosphorylated forms. In addition, phosphopeptides may show inefcient ionization or may be lost preferentially during handling by adsorption to metal or plastics. Thus, a large repertoire of techniques has been developed to enrich phosphoanalytes and improve detection sensitivity, particularly for samples of high complexity. Many of these make use of reactive chemistries for covalent coupling or afnity purication. The latter approach includes immobilized metal-afnity chromatography (IMAC), ion-exchangeHPLC, and antibody-based afnity chromatography. The IMAC method has been shown to facilitate the recovery, sequencing, and assignment of hundreds of phosphopeptides from complex protein samples, such as plant membrane proteins (for review, see the article by Jensen(120) ). 6.2 Lipoproteins CCC is the generic name for various liquidliquid partition chromatographic methods used without solid support matrices.(121) The stationary phase is retained in the column with the aid of gravity or centrifugal force. In this way, the system eliminates all complications arising from the solid support. Previous studies have shown that a mixture of HDL and LDL fractions prepared by ultracentrifugation could be separated by x-axis ultracentrifugation.(122) Recently, the complementary use
of CCC was attempted for the separation of three main classes of lipoproteins. Consequentely, the fractionation of HDLs, VLDLs, and LDLs may be performed by the combined use of polymer-phase CCC and hydroxyapatite chromatography without prior ultracentrifugation.(123) In the last decade, CE has emerged as a powerful separation technique for separation of plasma apolipoproteins.(124) By adding the surfactant SDS to the separation buffer, the main apolipoproteins of HDL (apoA-I and apoA-II) and LDL (apoB) may be separated in a single run in <12 min. The CE separation of VLDL apolipoproteins has also been studied. High-performance capillary isotachophoresis (ITP) of lipoproteins has been reported, which provided for rapid screening of lipoprotein abnormalities.(125)
COMPARISON WITH OTHER METHODS
To summarize, it is not surprising that HPLC techniques, given the wide versatility, relative ease of use, and high resolution, may be considered the most valuable tool for the characterization of virtually any hydrophobic protein. The great advantage of the HPLC method lies in the fact that it can be hyphenated on-line to ESI-MS to obtain intact mass data suitable for protein identication. The separation of each protein by HPLC is far superior to that possible with SDS-PAGE, and the identication of proteins is easy and quick by intact mass measurements, avoiding trypsin digestion and mass ngerprinting of proteins, which is often not sufcient for unequivocal protein identication because of the limited number of peptides obtained from these hydrophobic proteins. Our observations that the chromatographic behavior of the PSI antenna proteins in RP-HPLC is similar in all species investigated along with the strong correlation revealed between experimental retention times of the antenna proteins examined and their predicted values based on the overall hydrophobicity and polypeptide chain length mean that RP-HPLC could probably be used to provide accurate and reliable ngerprints of proteins prepared from different species, without resorting to protein identication by additional methods. A further benet derives from the fact that the peak area of chromatograms is proportional to the quantity of the separated sample, so the method may be suitable for estimating the stoichiometry of proteins. This provides an attractive alternative technique with which to monitor the subtle changes that often accompany physiological adaptations in terms of concentration of components and addition of chemical modications, such as phosphorylation, through the increase in intact molecular mass. In the particular case of thylakoid membrane, the ability to simultaneously identify and quantify a large number of proteins, such as
30
most of the components of PSI and PSII, will greatly facilitate investigation of stress-induced changes in the stoichiometry of the supramolecular complexes. New supports offer a good compromise between mechanical stability and nonspecic binding, as well as an excellent recovery of the protein loaded. Moreover, these matrices also have excellent chemical stability within the working range of biological separations. In this context, the use of a RP C-4 column, where the interaction of proteins with the matrix is very soft, has allowed a good separation and the complete recovery of proteins when applied to the separation of photosynthetic apparatus.(77) In this particular case, where the protein molecular masses are similar and the amino acid sequence is sufciently conserved, electrophoresis and immunoblotting appeared expensive and technically demanding. Moreover, for quantitative analysis, traditional approaches by SDS/PAGE are not only cumbersome but also rather ineffective for evaluating differences in the relative quantity of each component, unless time-consuming antibody titration is used.(126) Recent availability of high-resolution scanners and highly sensitive colorations and labeling approach (for example, through uorophore-linked CyDyes, which is at the basis of the success of high-performance differential gel electrophoresis using uorescent dyes (DIGE)) have partially helped overcoming this issue hampering broader diffusion of gel-based approaches.(62) However, recent introduction of quantitative approaches involving HPLC-MS makes it desirable to pursue quantitation through the chromatographic/mass spectrometric workow streamline, especially in the elds of proteomics and metabolomics. In Table 3 are summarized the advantages and disadvantages of separations performed by HPLC with other alternative separation systems such as SDS/PAGE, IEF, and CE. The number of reports dealing with the separation of membrane proteins by high-performance CE is still rather small. First results indicate that virtually all methods used for the separation of watersoluble proteins can also be applied to the separation of hydrophobic membrane proteins. Apart from the guidelines mentioned above, the know-how acquired
in high-performance CE and in the preparative freeow (ITP) of serum lipoproteins can be transferred to the separation of membrane proteins.(127) We have demonstrated that capillary zone electrophoresis (CZE) can be successfully applied for the complete resolution of the LHCII thylakoid membrane proteins by using the neutral detergent OD at a concentration lower than its critical micelle concentration in the electrolyte solution.(128) This method was revealed to be rapid and sensitive for identifying and determining quantitatively the several components of PSII, but the RP-HPLC method,(77) in addition to being rapid, simple, and precise, has proven to be effective at detecting differences in the protein components of LHCII isolated from different plants that might not be evidenced by denaturing SDS/PAGE, as in the case of many species (data not shown). In addition, the possibility of separating all protein components of the PSII major and minor antenna systems in samples not subjected to sucrose gradient ultracentrifugation, as in the case of injection of BBY, is expected to be advantageous for evaluating the relative content of the different protein components of PSII and their variation related to physiological adaptation to environmental conditions. The injection of BBY directly allows one to obtain an exact evaluation of the quantitative relationships between chlorophyll a/b binding present in PSII, avoiding fractionated separation, Coomassie brilliant blue staining, quantication by densitometry, and correction of the results according to the specic binding of Coomassie brilliant blue to isolated proteins, as required in SDS/PAGE. Using this method in screening photosynthetic mutants and plants adapted to different environmental conditions will help in the elucidation of the composition and supramolecular organization of LHCII and will possibly increase the understanding of the molecular mechanisms underlying the physiological adaptations. As shown in the application examples in Section 5, all HPLC methods developed for membrane protein separations offer the further advantage of analyzing the content of each HPLC peak by a multidimensional approach. A single peak observed in the RP-HPLC
Table 3 Comparison of the advantages and disadvantages of separations performed by HPLC and other methods
Parameter Analysis time Reproducibility Sample amount (L) Resolution Sensitivity Quantitative analysis Automation RP-HPLC 30 min Excellent 150 Good Nanograms Excellent Yes CZE 10 min Reasonable 15 Good Picograms Good Yes IEF Hours Operator dependent 1100 Excellent Micrograms Reasonable No SDS/PAGE 1824 h Operator dependent 1100 Excellent Micrograms Reasonable No BN-PAGE 1824 h Operator dependent 1100 Excellent Micrograms Reasonable No
31
separation of a protein is not an indication that the protein is highly pure because it is common for related substances to co-elute with the parent protein. The main peak can be isolated and reinjected into an alternative separation system such as an SDS/PAGE, IEF, CE, or dissimilar HPLC system, taking appropriate measures to avoid decomposition of the protein after collection from the HPLC system. If the HPLC solvent system is not compatible with the second separation system, or if suitable stability of the isolated main peak is not attainable, then the nonfractionated protein product can be subjected to one of the alternative separation procedures. On-line spectroscopic monitoring using diode-array detection can be of value in establishing peak purity in favorable cases, but usually protein spectra are too similar to provide much discrimination. The RP-HPLC/ESI-MS method holds several advantages over SDS/PAGE, the conventional technique for studying membrane proteins, including better protein separation, mass accuracy, speed, and efciency. Our study has shown that RP-HPLC/ESI-MS is an effective method for separating and characterizing the integral membrane proteins comprising the PSII major and minor antenna systems, both as isolated complexes by sucrose gradient ultracentrifugation and as the BBY grana membrane preparation directly. In accordance with the molecular genetic data reported in the literature, showing that higher plants have several Lhcb1 genes encoding different type I proteins for each species, two type I proteins of similar molecular mass have been resolved in spinach leaves. The experimental data are in good agreement with the molecular masses of the individual antenna proteins calculated on the basis of their nucleotide-derived amino acid sequences. In addition, the RP-HPLC/ESI-MS method allows the separation of protein constituents of the major and minor antenna systems, which are not resolved by conventional SDS/PAGE methods. Other advantages of RPHPLC/ESI-MS over SDS/PAGE include the accuracy in determining the molecular mass and the higher speed and efciency. However, recent advancements in the eld of gel-based approaches have made it possible to rely on alternative methods for separation of more hydrophobic proteins, mainly in their native conditions and as aggregates (for a recent review, the interested reader is referred to Helbig et al.(3) ). Nonetheless, these techniques are still rather underdiffused and require a specic technical expertise. The use of chromatographic methods offers the possibility of combining more techniques, and therefore, both SEC and RP-HPLC methods will be employed to provide high-resolution, quantitative data, in addition to various spectroscopic, electrophoretic, and immunochemical procedures for more specialized purposes. Conventional
electrophoretic techniques often do not directly add to the information obtained because HPLC frequently offers better resolution of related substances. In this respect, 2-D-HPLC approaches involving multiple dimension separation of protein species through chromatography have further expanded the role of chromatographic techniques in the protein-oritented area of investigations, giving birth to the so-called shotgun proteomics approach. Indeed, recently introduced MudPIT approaches have contributed to make HPLC one of the eligible strategies for protein separation, and also for the more hydrophobic fraction.(11) However, a key advantage of conventional electrophoretic techniques is the ability to detect virtually any protein, thereby ensuring that any unanticipated protein impurities would be detected. In developing the HPLC methods to be used in the battery of tests, one must also consider the information obtained from the other techniques and seek to understand the relationship between these sets of information. The objective of such an assessment is to obtain the maximum amount of relevant information consistent with an efcient use of analytical resources. Concerning lipoproteins separation, it was reported in Section 6 that by using CE, high separation efciencies and short analysis times for lipoproteins can be achieved. CE exhibits advantages over HPLC for lipoprotein analysis: CE separations are faster and do not require the use of expensive columns, and CE does not suffer from slow mass transfer rates, which lead to band broadening in HPLC separations of apolipoproteins. The main advantage of CE over conventional electrophoresis is speed and instrumental format, which eliminates the need for labor-intensive steps such as gel preparation and staining. Structural studies on proteins depend on an investigators ability to isolate and purify the protein. In many cases, protein isolation is a trivial matter, but purication to homogeneity is a lengthy process. Traditionally, proteins have been puried by a combination of precipitation; open-column chromatography, including SEC and ion-exchange chromatography; ultracentrifugation; and electrophoresis. HPLC has become increasingly important in the purication and analysis of proteins.(129) Brilliant strategies targeting membrane protein structures through X-ray crystallography have helped to accumulate knowledge on membrane proteins. Indeed, structural information is as much as fundamental as qualitative and quantitative results that can be obtained through HPLC (and MS) analyses. These information are relevant not only to understand conformational variations but also to build up reliable models, which reect the functional role of a specic membrane protein or complex, as in the case of plant light-harvesting complex
32
Figure 14 Stereo diagram of the temperature factor distribution in the structure of spinach LHCII (PDB code 1RWT). The color gradient ranges from blue (lower-temperature factors, rigid) to red (higher-temperature factors, exible). The only part of the structure with a high-temperature factor, indicating signicant exibility, is the protruding part of Neo (arrows). The asterisk indicates Chl 2. Detergent, lipids, and phytyl chains have been omitted for clarity. The approximate position of the lipid bilayer is indicated in gray. (Reproduced from Ref. 130. Nature Publishing Group, 2009.) in spinach (Figure 14).(130) In these very cases, HPLC could be extremely helpful in preliminary purication (and purity assessment steps), which are fundamental to guarantee optimal crystallization and subsequent structural analyses.(130) Finally, recent trends in protein-oriented studies aim at determining quantitative and posttranslational modication changes in biological samples. In this respect, chromatography appears to be the most suitable analytical method to be performed before nal MS analysis. CE CHAPS Capillary Electrophoresis 3-[(3-Cholamidopropyl) Dimethylammonio]-lpropanesulfonate Coronary Heart Disease Collision-Induced Dissociation ) Clear Native Clear Native Polyacrylamide Gel Electrophoresis Controlled Pore Glass Cetyltrimethylammonium Bromide Polyacrylamide Gel Electrophoresis Capillary Zone Electrophoresis n-Dodecyl -D-maltoside Two-Dimensional Electophoresis Two-Dimensional High-Performance Liquid Chromatography Two-Dimensional Isoelectrofocusing/Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis Differential in-Gel Electrophoresis Dodecil Maltoside n-Dodecyl--D-maltoside Dithiothreitol Ethylenediaminetetraacetic Acid Ethylene Glycol Bis(-Aminoethyl
CHD CID CN CN-PAGE CPG CTAB-PAGE
ACKNOWLEDGMENTS
I am grateful to former and present members of the research group who have contributed to much of the work presented in this review and to Dr Anna Maria Timperio and Dr Sara Rinalducci for their valuable assistance in the preparation of the manuscript. This work was supported by CE Project CIPA CT93 0202 and COST Contract ERB IC15CT 980126.
CZE DDM 2-DE 2-D-HPLC
2-D-IEF/SDS-PAGE
ABBREVIATIONS AND ACRONYMS

AIDS BLM BN BN-PAGE CCC Acquired Immune Deciency Syndrome Perfusion Planar Lipid Membrane Blue Native Blue Native Polyacrylamide Gel Electrophoresis Countercurrent Chromatography DIGE DM -DM DTT EDTA EGTA
33
Phenylmethanesulfonyl Fluoride Polystyrene/divinylbenzene Photosystem I Photosystem II Reversed-Phase Reversed-Phase High-Performance Liquid Chromatography Room Temperature Soluble Cd4 Strong Cation-Exchange Strong Cation-Exchange High-Performance Liquid Chromatography Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis Sodium Dodecyl Sulfate Size-Exclusion High-Performance Liquid Chromatography Size-Exclusion Chromatography ) Triuoroacetic Acid Total Ion Current Time-of-Flight Ultraviolet Very Low-Density Lipoprotein
ELISA ESI ESI-MS FA FFE FPLC GE GPC HDL HEPES
HIV HLP HPLC HPLC/MS
IA ICAT ID IEF IMAC ITP iTRAQ LC LDL LHCI LHCII MAb MALDI MES MS MudPIT NADP OD
Ether)-N ,N ,N ,N -Tetraacetic Acid Enzyme-Linked Immunosorbent Assay Electrospray Ionization Electrospray Ionization Mass Spectrometry Formic Acid Free Flow Electrophoresis ) Fast Protein Liquid Chromatography Gel Electrophoresis Gel Permeation Chromatography High-Density Lipoprotein N -(2Hydroxyethyl)Piperazine-N (2-Ethanesulfonic Acid) Human Immunodeciency Virus Hyperlipoproteinemic High-Performance Liquid Chromatography High-Performance Liquid Chromatography/mass Spectrometry Immunoafnity Isotope-Coded Afnity Tagging Internal Diameter Isoelectric Focusing Immobilized Metal-Afnity Chromatography Isotachophoresis Isotope Tagging for Relative and Absolute Quantitation Liquid Chromatography Low-Density Lipoprotein Light-Harvesting Complex of Photosystem I Light-Harvesting Complex of Photosystem II Monoclonal Antibody Matrix-Assisted Laser Desorption/ionization Morpholinoethanesulfonic Acid Mass Spectrometry Multidimensional Protein Identication Technology Nicotinamide Adenine Dinucleotide Phosphate n-Octyl -D-glucopyranoside
PMSF PS-DVB PSI PSII RP RP-HPLC
RT sCD4 SCX SCX-HPLC
SDS/PAGE
SDS SE-HPLC
SEC TFA TIC TOF UV VLDL
RELATED ARTICLES
Biomolecules Analysis (Volume 1) High-performance Liquid Chromatography of Biological Macromolecules Mass Spectrometry of Biological Molecules Carbohydrate Analysis (Volume 1) Glycoprotein Analysis: General Methods Clinical Chemistry (Volume 2) Biochemical Markers of Acute Coronary Syndromes Capillary Electrophoresis in Clinical Chemistry Lipid Analysis for Important Clinical Conditions Phospholipids of Plasma Lipoproteins, Red Blood Cells and Atheroma, Analysis of Serum Proteins Particle Size Analysis (Volume 6) Centrifugation in Particle Size Analysis
34
Peptides and Proteins (Volume 7) Capillary Electrophoresis in Peptide and Protein Analysis, Detection Modes for Capillary Electrophoresis of Proteins and Glycoproteins Capillary Electrophoresis/Mass Spectrometry in Peptide and Protein Analysis Gel Electrophoresis in Protein and Peptide Analysis Miniaturization of High Performance Liquid Chromatography Separations and Equipment in Peptide and Protein Analysis High-performance Liquid Chromatography/Mass Spectrometry in Peptide and Protein Analysis Reversed-phase High-performance Liquid Chromatography in Peptide and Protein Analysis Liquid Chromatography (Volume 13) Liquid Chromatography: Introduction Afnity Chromatography Capillary Electrophoresis Gradient Elution Chromatography Ion Chromatography Reversed Phase Liquid Chromatography Mass Spectrometry (Volume 13) Liquid Chromatography/Mass Spectrometry
14. 9.
W. Zhang, G. Zhou, Y. Zhao, M.A. White, Y. Zhao, Afnity Enrichment of Plasma Membrane for Proteomics Analysis, Electrophoresis, 24(16), 28552863 (2003). D. Ghosh, R.C. Beavis, J.A. Wilkins, The Identication and Characterization of Membranome Components, J. Proteome Res., 7(4), 15721583 (2008). M.P. Washburn, D. Wolters, J.R. Yates 3rd. Large-scale Analysis of the Yeast Proteome by Multidimensional Protein Identication Technology, Nat. Biotechnol., 19(3), 242247 (2001). C. Huber, A.M. Timperio, H. Toll, L. Zolla, Liquidchromatography-mass Spectrometry of Thylakoid Membrane Proteins, Methods Mol. Biol., 492, 113130 (2009). P.E. Gustavsson, P.O. Larsson, Supporting Material for Afnity Chromatography, Handbook of Afnity Chromatography, Chromatographic Science Series, 2nd edition, Taylor & Francis Group, Broken Sound Parkway, NW, 1535, Vol. 92, 2006. S.H. Lin, G. Guidotti, Purication of Membrane Proteins, Methods Enzymol., 463, 619629 (2009). K.K. Tetala, T.A. van Beek, Bioafnity Chromatography on Monolithic Supports, J. Sep. Sci., 33(3), 422338 (2010). K. Mondal, M.N. Gupta, The Afnity Concept in Bioseparation: Evolving Paradigms and Expanding Range of Applications, Biomol. Eng., 23(23), 5976 (2006). R.L. Gundry, M.Y. White, C.I. Murray, L.A. Kane, Q. Fu, B.A. Stanley, J.E. Van Eyk, Preparation of Proteins and Peptides for Mass Spectrometry Analysis in a Bottom-up Proteomics Workow, Curr. Protoc. Mol. Biol., 88, 10.25.110.25.23 (2009). P. Lundahl, Q. Yang, Liposome Chromatography: Liposomes Immobilized in Gel Beads as a Stationary Phase for Aqueous Column Chromatography, J. Chromatogr., 544(2), 283304 (1991). P. Lundahl, C.M. Zeng, C. Lagerquist Hagglund, I. Gottschalk, E. Greijer, Chromatographic Approaches to Liposomes, Proteoliposomes and Biomembrane Vesicles, J. Chromatogr., B Biomed. Sci. Appl., 722(12), 103120 (1999). C.C. Wu, M.J. MacCoss, Shotgun Proteomics: Tools for the Analysis of Complex Biological Systems, Curr. Opin. Mol. Ther., 4(3), 242250 (2002). C. Delahunty, J.R. Yates 3rd. Protein Identication Using 2D-LC-MS/MS, Methods, 35(3), 248255 (2005). L. Florens, M.P. Washburn, Proteomic Analysis by Multidimensional Protein Identication Technology, Methods Mol. Biol., 328, 159175 (2006).
10.
11.
12.
13.
REFERENCES
1. P.G. Sadowski, A.J. Groen, P. Dupree, K.S. Lilley, Subcellular Localization of Membrane Proteins, Proteomics, 8(19), 39914011 (2008). D. Plewczynski, L. Rychlewski, Meta-basic Estimates the Size of Druggable Human Genome, J. Mol. Model., 15(6), 695699 (2009). A.O. Helbig, A.J. Heck, M. Slijper, Exploring the Membrane ProteomeChallenges and Analytical Strategies, J. Proteomics., 73(5), 868878 (2010). T. Rabilloud, M. Chevallet, S. Luche, C. Lelong. Twodimensional Gel Electrophoresis in Proteomics: Past, Present and Future. J. Proteomics, (2010). DOI: 10.1016/j.jprot.2010.05.016 O.P. Dancette, J.L. Taboureau, E. Tournier, C. Charcosset, P. Blond, Purication of Immunoglobulins G by Protein A/G Afnity Membrane Chromatography, J. Chromatogr., B Biomed. Sci. Appl., 723(12), 6168 (1999). E.P. Carpenter, K. Beis, A.D. Cameron, S. Iwata, Overcoming the Challenges of Membrane Protein Crystallography, Curr. Opin. Struct. Biol., 18(5), 581586 (2008). D. Josic, J.G. Clifton, S. Kovac, D.C. Hixson, Membrane Proteins as Diagnostic Biomarkers and Targets for New Therapies, Curr. Opin. Mol. Ther., 10(2), 116123 (2008). A.M. Seddon, P. Curnow, P.J. Booth, Membrane Proteins, Lipids and Detergents: Not Just a Soap Opera, Biochim. Biophys. Acta, 1666(12), 105117 (2004).
15.
16.
2.
3.
17.
4.
18.
5.
19.
6.
20.
7.
21. 22.
8.
35
H.Y. Sun, S.F. Chen, M.D. Lai, T.T. Chang, T.L. Chen, P.Y. Li, D.B. Shieh, K.C. Young, Comparative Proteomic Proling of Plasma Very-low-density and Lowdensity Lipoproteins, Clin. Chim. Acta, 411(56), 336344 (2010). T. Sata, D.L. Estrich, P.D.S. Wood, L.W. Kinsell, Evaluation of Gel Chromatography for Plasma Lipoprotein Fractionation, J. Lipid Res., 11(4), 331340 (1970). W. Marz, R. Skeimer, H. Scharnagi, U.B. Seiffert, W. Gross, Fast Lipoprotein Chromatography: New Method of Analysis for Plasma Lipoproteins, Clin. Chem., 39(11), 22762281 (1993). P.M. Clifton, A.M. MacKinnon, P.J. Barter, Gel permeation Chromatography of Lipoproteins, J. Chromatogr., 414, 2534 (1987). H.M. Wilson, B.A. Grifn, C. Watt, E.R. Skinner, The Isolation and Characterization of High-densitylipoprotein Subfractions Containing Apolipoproteins E from Human Plasma, Biochem. J., 284(2), 477482 (1992). T. OBr` en, J. Buithieu, T.T. Nguyen, L. Klein, N. Bren, M. Wentworth, B.J. Hallaway, B.A. Kottke, Separation of High-density Lipoproteins into Apolipoprotein epoor and Apolipoprotein e-rich Subfractions by Fast Protein Liquid Chromatography using a Heparin Afnity Column, J. Chromatogr., 613(2), 239246 (1993). B. Vedie, I. Myara, M.A. Pech, I.C. Maziere, C. Maziere, A. Capran` N. Moatti, Fractionation of Charge-modied , Low Density Lipoproteins by Fast Protein Liquid Chromatography, J. Lipid Res., 32(8), 13591369 (1991). J.A.K. Harmony, E.I. Cordes, Interaction of Human Plasma Low Density Lipoprotein with Concanavalin A and with Ricin, J. Biol. Chem., 250(22), 86148617 (1975). D.A. Blasiole, A.T. Oler, A.D. Attie, Regulation of ApoB Secretion by the Low Density Lipoprotein Receptor Requires Exit from the Endoplasmic Reticulum and Interaction with ApoE or ApoB, J. Biol. Chem., 283(17), 1137411381 (2008). P.M. Young, T.M. Boehrn, Rapid and Efcient Separation of Apolipoproteins, J. Chromatogr., A, 31, 7985 (1984). T.A. Hughes, M.A. Moore, P. Neame, M.F. Medley, B.H. Cheung, Rapid Quantitative Apolipoprotein Analysis by Gradient Ultracentrifugation and Reversedphase High Performance Liquid Chromatography, J. Lipid Res., 29(3), 363376 (1988). M.E. Campanella, H. Chu, P.S. Low, Assembly and Regulation of a Glycolytic Enzyme Complex on the Human Erythrocyte Membrane, Proc. Natl. Acad. Sci. U.S.A., 102(7), 24022407 (2005).
23. J. Han, K.L. Schey, Proteolysis and Mass Spectrometric Analysis of an Integral Membrane: Aquaporin 0, J. Proteome Res., 3(4), 807812 (2004). 24. H. Zhong, S.L. Marcus, L. Li, Microwave-assisted Acid Hydrolysis of Proteins Combined with Liquid Chromatography MALDI MS/MS for Protein Identication, J. Am. Soc. Mass Spectrom., 16(4), 471481 (2005). 25. D.K. Han, J. Eng, H. Zhou, R. Aebersold, Quantitative Proling of Differentiation-induced Microsomal Proteins Using Isotope-coded Afnity Tags and Mass Spectrometry, Nat. Biotechnol., 19(10), 946951 (2001). 26. P. Bailon, M. Nachman-Clewner, C.L. Spence, G.K. Ehrlich, Receptor-afnity Chromatography, Handbook of Afnity Chromatography, Chromatographic Science Series, 2nd edition, Taylor & Francis Group, Broken Sound Parkway, NW, 435461, Vol. 92, 2006. 27. D. Josic, J. Reusch, K. Loster, O. Baum, W. Reutter, High-performance Membrane Chromatography of Serum and Plasma Membrane Proteins, J. Chromatogr., 590(1), 5976 (1992). 28. J.T. Skare, E.S. Shang, D.M. Foley, D.R. Blanco, C. Champion, T. Mirzabekov, Y. Sokolov, B.L. Kagan, J.N. Miller, M.A. Lovett, Virulent Strain Associated Outer Membrane Proteins of Borrelia burgdorferi , J. Clin. Invest., 96(5), 23802392 (1995). 29. T.A. Mirzabrkov, A.Y. Silberstein, B.L. Kagan, Use of Planar Lipid Bilayer Membranes for Rapid Screening of Membrane Active Compounds, Methods Enzymol., 271, 661674 (1996). 30. J.T. Skare, T.A. Mirzabekov, E.S. Shang, D.R. Blanco, H. Erdjument-Bromage, J. Bunikis, S. Bergstrom, P. Tempst, B.L. Kagan, J.N. Miller, M.A. Lovett, The Oms66 (p66) Protein is a Borrelia burgdorferi Porin, Infect. Immun., 65(9), 36543661 (1997). 31. T. Mirzabekov, M.C. Lin, W.L. Yuan, P.J. Marshall, M. Carman, K. Tomaselli, I. Lieberburg, B.L. Kagan, Channel Formation in Planar Lipid Bilayers by a Neurotoxic Fragment of the Beta-amyloid Peptide, Biochem. Biophys. Res. Commun., 202(2), 11421148 (1994). 32. A.M. Bielik, J. Zaia, Historical Overview of Glycoanalysis, Methods Mol. Biol., 600, 930 (2010). 33. S.P. McCormick, Lipoprotein(a): Biology and Clinical Importance, Clin. Biochem. Rev., 25(1), 6980 (2004). 34. L. Ago ton-Coldea, L.D. Rusu, D. Zdrenghea, M.L. s Rusu, D. Pop, A. Craciun, L. Poanta, M. Gatfosse, S. Rosenstingl, T. Mocan, Lipoprotein (a) and Lipid and Non-lipid Risk Factors in Coronaries Risk Assessment, J. Intern. Med., 46(2), 137144 (2008). 35. Y. Shibusawa, Lipoproteins: Comparison of Different Separation Strategies, J. Chromatogr., B, 699, 419437 (1997).
36.
37.
38.
39.
40.
41.
42.
43.
44.
45.
46.
47.
36
48. L.A. Huber, K. Pfaller, I. Vietor, Organelle Proteomics: Implications for Subcellular Fractionation in Proteomics, Circ. Res., 92(9), 962968 (2003). 49. C. Pasquali, I. Fialka, L.A. Huber, Subcellular Fractionation, Electromigrationanalysis and Mapping of Organelles, J. Chromatogr., B Biomed. Sci. Appl., 722(12), 89102 (1999). 50. Z.W. Lai, D.L. Steer, A.I. Smith, Membrane Proteomics: the Development of Diagnostics Based on Protein Shedding, Curr. Opin. Mol. Ther., 11(6), 623631 (2009). 51. S.J. Cordwell, T.E. Thingholm, Technologies for Plasma Membrane Proteomics, Proteomics, 10(4), 611627 (2010). 52. D.E. Macfarlane, Use of Benzyldimethyl-n-hexadecylammonium Chloride (16-BAC), a Cationic Detergent, in an Acidic Polyacrylamide Gel Electrophoresis System to Detect Base Labile Protein Methylation in Intact Cells, Anal. Biochem., 132(2), 231235 (1983). 53. B. Wenge, H. Bonisch, J. Grabitzki, G. Lochnit, B. Schmitz, M.H. Ahrend, Separation of Membrane Proteins by Two-dimensional Electrophoresis Using Cationic Rehydrated Strips, Electrophoresis, 29, 15111517 (2008). 54. S. Helling, E. Schmitt, C. Joppich, T. Schulenborg, S. Mullner, S. Felske-Muller, 2-D Differential Membrane Proteome Analysis of Scarce Protein Samples, Proteomics, 6, 45064513 (2006). 55. H. Schagger, G. von Jagow, Blue Native Electrophoresis for Isolation of Membrane Protein Complexes in Enzymatically Active Form, Anal. Biochem., 199, 223231 (1991). 56. H. Schagger, W.A. Cramer, G. von Jagow, Analysis of Molecular Masses and Oligomeric States of Protein Complexes by Blue Native Electrophoresis and Isolation of Membrane Protein Complexes by Two-dimensional Native Electrophoresis, Anal. Biochem., 217, 220230 (1994). 57. H. Schagger, K. Pfeiffer, Supercomplexes in the Respiratory Chains of Yeast and Mammalian Mitochondria, EMBO J., 19, 17771783 (2000). 58. B. Kaboord, M. Perr, Isolation of Proteins and Protein Complexes by Immunoprecipitation, Methods Mol. Biol., 424, 349364 (2008). 59. M. Monti, M. Cozzolino, F. Cozzolino, G. Vitiello, R. Tedesco, A. Flagiello, P. Pucci, Puzzle of Protein Complexes in vivo: a Present and Future Challenge for Functional Proteomics, Expert Rev. Proteomics, 6(2), 159169 (2009). 60. D. Claeys, K. Geering, B.J. Meyer, Two-dimensional Blue Native/Sodium Dodecyl Sulfate Gel Electrophoresis for Analysis of Multimeric Proteins in Platelets, Electrophoresis, 26(6), 11891199 (2005). 67. 64. 61.
R.A. van Gestel, W.W. van Solinge, H.W. van der Toorn, G. Rijksen, A.J. Heck, R. van Wijk, M. Slijper, Quantitative Erythrocyte Membrane Proteome Analysis with Blue-Native/SDS PAGE, J. Proteomics, 73(3), 456465 (2010). I. Wittig, M. Karas, H. Schagger, High Resolution Clear Native Electrophoresis for In-Gel Functional Assays and Fluorescence Studies of Membrane Protein Complexes, Mol. Cell Proteomics, 6, 12151225 (2007). G. Mitulovic, K. Mechtler, HPLC Techniques for Proteomics Analysisa Short Overview of Latest Developments, Brief. Funct. Genomic. Proteomics, 5(4), 249260 (2006). I. Sekigawa, J.E. Groopmen, J.D. Allan, K. Ikeuchi, G. Bibereld, K. Takatsuki, R.A. Byrn, Characterization of Autoantibodies to the CD4 Molecule in Human Immunodeciency Virus Infection, Clin. Immunol. Immunopathol., 58(1), 145153 (1991). C. Jones, A. Patel, S. Grifn, J. Martin, P. Young, K. ODonnell, C. Silverman, T. Porter, I. Chaiken, Current Trends in Molecular Recognition and Bioseparation, J. Chromatogr., A, 707, 322 (1995). P.A. Well, B. Beiderman, R.L. Garlick, S.B. Lyle, J.P. Martin Jr, J.T. Rerberg, H.F. Meyer, S.L. Henderson, F.M. Eckenrode, Large-scale Immunoafnity Purication of Recombinant Soluble Human Antigen CD4 from Escherichia coli Cells, Biotechnol. Appl. Biochem., 18, 341357 (1993). B.S. Welinder, H.F. Sorensen, B. Hansen, Highperformance Liquid Chromatographic Separation of Membrane Proteins Isolated from Erythrocyte Ghosts, J. Chromatogr., 462, 255268 (1989). E.M. Pasini, M. Kirkegaard, P. Mortensen, H.U. Lutz, A.W. Thomas, M. Mann, In-depth Analysis of the Membrane and Cytosolic Proteome of Red Blood Cells, Blood, 108(3), 791801 (2006). G.M. DAmici, S. Rinalducci, L. Zolla, Proteomic Analysis of RBC Membrane Protein Degradation During Blood Storage, J. Proteome Res., 6, 32423255 (2007). A. Shevchenko, M. Wilm, O. Vorm, M. Mann, Mass Spectrometric Sequencing of Proteins Silver-stained Polyacrylamide Gels, Anal. Chem., 68, 850858 (1996). J. Barber, Imperial College, London. B. Hankamer, J. Barber, E.J. Boekema, Structure and Membrane Organization of Photosystem II in Green Plants, Annu. Rev. Plant Physiol. Plant Mol. Biol., 48, 641671 (1997). B.R. Green, D. Shen, R. Aebersold, E. Pichersky, Identication of the Polypeptides of the Major Lightharvesting Complex of Photosystem II (LHCII) with Their Genes in Tomato, FEBS Lett., 305(1), 1822 (1992).
62.
63.
65.
66.
68.
69.
70.
71. 72.
73.
37
P. Dainese, R. Bassi, Subunit Stoichiometry of the Chloroplast Photosystem II Antenna System and Aggregation State of the Component Chlorophyll a/b Binding Proteins, J. Biol. Chem., 266(13), 81368142 (1991). N. Krauss, W. Hinrichs, I. Witt, P. Fromme, M. Pritzkow, Z. Dauter, C. Betzel, K.S. Wilson, H.T. Witt, W. Saenger, Three-dimensional Structure of System I of Photosynthesis at 6 A Resolution, Nature (London), 361, 326331 (1993). P.R. Chitnis, Q. Xu, V.P. Chitnis, R. Nechushtai, Function and Organization of Photosystem Polypeptides, Photosynth. Res., 44(1), 2340 (1995). A.M. Timperio, C.G. Huber, L. Zolla, Separation and Identication of the Light Harvesting Proteins Contained in Grana and Stroma Thylakoid Membrane Fractions, J. Chromatogr., A., 1040(1), 7381 (2004). W. Walcher, H. Oberacher, S. Troiani, G. Holzl, P. Oefner, L. Zolla, C.G. Huber, Monolithic Capillary Columns for Liquid Chromatography-electrospray Ionization Mass Spectrometry in Proteomic and Genomic Research, J. Chromatogr., B Anal. Technol. Biomed. Life Sci., 782(12), 111125 (2002). U. Matsumoto, H. Nakayama, Y. Shibusawa, T. Niimura, Separation of Human Serum Lipoproteins into Three Major Classes by Hydroxyapatite Chromatography, J. Chromatogr., 566(1), 6776 (1991). C.G. Huber, A. Premstaller, W. Xiao, H. Oberacher, G.K. Bonn, P.J. Oefner, Mutation Detection by Capillary Denaturing High-performance Liquid Chromatography Using Monolithic Columns, J. Biochem. Biophys. Methods, 47(12), 519 (2001). L. Zolla, A.M. Timperio, High Performance Liquid Chromatography-electrospray Mass Spectrometry for the Simultaneous Resolution and Identication of Intrinsic Thylakoid Membrane Proteins, Proteins, 41(3), 398406 (2000). J. Sharma, M. Panico, J. Barber, H.R. Morris, Purication and Determination of Intact Molecular Mass by Electrospray Ionization Mass Spectrometry of the Photosystem II Reaction Center Subunits, J. Biol. Chem., 272(52), 3315333157 (1997). A.M. Timperio, L. Zolla, Investigation of the Lateral Light-induced Migration of Photosystem II Lightharvesting Proteins by Nano-high Performance Liquid Chromatography Electrospray Ionization Mass Spectrometry, J. Biol. Chem., 280(32), 2885828866 (2005). I. Hara, M. Okazaki, High-performance Liquid Chromatography of Serum Lipoproteins, Methods Enzymol., 129, 5778 (1986). E. Koller, I. Volf, A. Gurvitz, F. Koller, Modied Lowdensity Lipoproteins and High-density Lipoproteins.
74. S. Jansson, The Light-harvesting Chlorophyll a/b Binding Proteins, Biochim. Biophys. Acta, 1184, 119 (1994). 75. R. Bassi, F. Rigoni, R. Barbato, G.M. Giacometti, Lightharvesting Chlorophyll a/b Protein (LHCI) Populations in Phosphorylated Membranes, Biochim. Biophys. Acta, 936, 2938 (1988). 76. L. Zolla, M. Bianchetti, A.M. Timperio, D. Corradini, Rapid Resolution by Reversed-phase High-performance Liquid Chromatography of the Thylakoid Membrane Proteins of the Photosystem II Light-harvesting Complex, J. Chromatogr., A, 779, 131138 (1997). 77. D.A. Berthold, G.T. Babcock, C.A. Yocum, A Highly Resolved Oxygen-evolving Photosystem II Preparation from Spinach Thylakoid Membranes, FEBS Lett., 134, 231234 (1981). 78. R.J. Porra, W.A. Thompson, P.E. Kriedemann, Determination of Accurate Extinction Coefcients and Simultaneous Equations for Assaying Chlorophyll a and b Extracted with Four Different Solvents: Verication of the Concentration of Chlorophyll Standards by Atomic Absorption Spectroscopy, Biochim. Biophys. Acta, 975,, 384394 (1989). 79. R. Bassi, P. Dainese, A Supramolecular Light-harvesting Complex from Chloroplast Photosystem-II Membranes, Eur. J. Biochem., 204, 317326 (1992). 80. L. Zolla, A.M. Timperio, M.G. Testi, M. Bianchetti, R. Bassi, D. Corradini, Isolation, Characterization and Subunits Stoichiometry of Chloroplast Photosystem II Antenna of Spinach by Reversed-phase Liquid Chromatography, Photosynth. Res., 63, 281290 (1999). 81. H. Schagger, G. von Jagow, Tricine-sodium Dodecyl Sulfate-polyacrylamide Gel Electrophoresis for the Separation of Proteins in the Range 1 to 100 kDa, Anal. Biochem., 166, 368379 (1987). 82. M.L. Di Paolo, A. Peruffo Dal Belin, R. Bassi, Immunological Studies on Chlorophyll a/b Proteins and Their Location in Chloroplast Membrane Domains, Planta, 191, 275286 (1990). 83. R. Bassi, F. Rigoni, G.M. Giacometti, Chlorophyll Binding Proteins with Antenna Function in Higher Plants and Green Algae, Photochem. Photobiol., 52, 11871206 (1990). 84. D. Corradini, C.G. Huber, A.M. Timperio, L. Zolla, Resolution and Identication of the Protein Components of the Photosystem II Antenna System of Higher Plants by Reversed-phase Liquid Chromatography with Electrospray-Mass Spectrometric Detection, J. Chromatogr., A., 886(12), 111121 (2000). 85. M. Sigrist, L.A. Staehelin, Identication of Type 1 and Type 2 Light-harvesting Chlorophyll a/b-binding Proteins Using Monospecic Antibodies, Biochim. Biophys. Acta, 1098, 191200 (1992).
86.
87.
88.
89.
90.
91.
92.
93.
94.
95.
96.
97.
38
From Investigation Tools to Real in vivo Players, Pathophysiol. Haemost. Thromb., 35(34), 322345 (2006). 98. A. Carpenter, W.C. Purdy, Preparation of Heparinglyceryl Controlled-pore Glass Afnity Media for the Separation of alpha- and beta-lipoproteins, J. Chromatogr., 573(1), 132135 (1992). 99. G. Steiner, Triglyceride-rich Lipoproteins and Atherosclerosis, from Fast to Feast, Ann. Med., 25(5), 431451 (1993). 100. I.J. Goldberg, W.D. Wagner, L. Pang, L. Paka, L.K. Curtiss, J.K. DeLozier, G.S. Shelness, C.S.H. Young, S. Pillarisetti, The NH2 -terminal Region of Apolipoprotein B is Sufcient for Lipoprotein Association with Glycosaminoglycans, J. Biol. Chem., 273, 3535535361 (1998). 101. R.W. James, A. Proudfoot, D. Pometta, Immunoafnity Fractionation of High-density Lipoprotein Subclasses 2 and 3 Using Anti-apolipoprotein A-I and A-II Immunosorbent Gels, Biochim. Biophys. Acta, 1002(3), 292301 (1989). 102. E. Koren, P. Alaupovic, D.M. Lee, N. Dashti, H.U. Kloer, G. Wen, Selective Isolation of Human Plasma Low-density Lipoprotein Particles Containing Apolipoproteins B and E by use of a Monoclonal Antibody to Apolipoprotein B, Biochemistry, 26(10), 27342740 (1987). 103. M.C. Cheung, Methods for Separation of Apolipoproteins, Methods Enzymol., 129, 130151 (1986). 104. C. Edeistein, in Analysis of Oils, Fats and Lipoproteins, ed. E.G. Perkins, American Oil Chemists Society, Champaign, IL, 589603, 1991. 105. M. Okazaki, M. Kinoshita, I. Hara, Improved Highperformance Liquid Chromatographic Method for the Determination of Apolipoproteins in Serum High-density Lipoproteins, J. Chromatogr., 430(1), 135142 (1988). 106. G.L. Mill, P.A. Lane, P.K. Weech, A Guide Book to Lipoprotein Techniques: Techniques in Biochemistry and Molecular Biology, Elsevier, Amsterdam, Vol. 14, 1984. 107. H. Mezdour, V. Clavey, I. Kora, M. Kofgan, A. Barkia, J.C. Fruchart, Anion-exchange Fast Protein Liquid Chromatographic Characterization and Purication of Apolipoproteins A-I, A-II, C-I, C-II, C-III0, C-III1, C-III2 and E from Human Plasma, J. Chromatogr., 414(1), 3545 (1987). 108. G. Hocke, H. Kaffarnik, G. Minscher, A. Steinemetz, Purication of Human Serum Amyloid A by Anion-exchange Fast Protein Liquid Chromatography, J. Chromatogr., 526(1), 203209 (1990). 109. R. Weinberg, C. Patton, B. DaGue, Analytic and Preparative Separation of Apolipoproteins A-I, A-II, and A-IV by Reverse Phase High Pressure Liquid Chromatography, J. Lipid Res., 29(6), 819824 (1988).
110. B. Meyer, E. Kecorius, P. Barter, N. Fidge, T. Tetaz, Application of Reversed-phase High-performance Liquid Chromatography to the Separation of Apolipoproteins A-IV, A-I and E from Rat High-density Lipoprotein, J. Chromatogr., A, 540, 386391 (1991). 111. A.J. Groen, K.S. Lilley, Proteomics of Total Membranes and Subcellular Membranes, Expert Rev Proteomics, 7(6), 867878 (2010). 112. M.L. Zeidel, S. Nielsen, B.L. Smith, S.V. Ambudkar, A.B. Maunsbach, P. Agre, Ultrastructure, Pharmacological Inhibition and Transport Selectivity of Aquaporin Channel-forming Integral Membrane Protein in Proteoliposomes, Biochemistry, 33, 16061611 (1994). 113. J.P. Whitelegge, B.C. Gundersen, K.F. Faull, Electrospray-ionization Mass Spectrometry of Intact Intrinsic Membrane Proteins, Protein Sci., 7, 14231430 (1998). 114. L. Zolla, C.H. Huber, A.M. Timperio, M. Bianchetti, D. Corradini, Use of RPHPLC with Electrospray Mass Spectrometric Detection for the Resolution and Identication of the Protein Components of PSII of Wheat, in Photosynthesis: Mechanisms and Effects, ed. L. Garab, Kluwer, Dordrecht, 43774380, 1998. 115. W. Hancock, A. Apffel, J. Chakel, C. Souders, T.M. Timkulu, E. Pungor Jr, A.W. Guzzetta, Reversedphase Peptide Mapping of Glycoproteins Using Liquid Chromatography/Electrospray Lonization Mass Spectrometry, Methods Enzymol., 271, 403427 (1996). 116. J. Wen, T. Arakawa, J.S. Philo, Size-exclusion Chromatography with on-line Light-scattering, Absorbance, and Refractive Lndex Detectors for Studying Proteins and Their Lnteractions, Anal. Biochem., 2401, 155166 (1996). 117. U. Jonsson, L. Fagerstam, B. Ivarsson, B. Johnsson, R. Karlsson, K. Lundh, S. Lofas, B. Persson, H. Roos, I. Ronnberg, Real-time Biospecic Interaction Analysis Using Surface Plasmon Resonance and Sensor Chip Technology, Biotechniques, 11(5), 620627 (1991). 118. A. Bachi, T. Bonaldi, Quantitative Proteomics as a New Piece of the Systems Biology Puzzle, J. Proteomics, 71(3), 357367 (2008). 119. E.S. Witze, W.M. Old, K.A. Resing, N.G. Ahn, Mapping Protein Post-translational Modications with Mass Spectrometry, Nat. Methods., 4(10), 798806 (2007). 120. O.N. Jensen, Modication-specic Proteomics: Characterization of Post-translational Modications by Mass Spectrometry, Curr. Opin. Chem. Biol., 8(1), 3341 (2004). 121. Y. Ito, in Counter-current Chromatography: Theory and Practice, eds. N.B. Mandava, Y. Ito, Marcel Dekker, New York, 1988. 122. Y. Shibusawa, Y. Ito, K. Ikewaki, D.J. Rader, B. Brewer, Counter-current Chromatography of Lipoproteins with a
39
Membranes from Maize Mesophyll and Bundle Sheath Chloroplasts, Eur. J. Biochem., 233(3), 709719 (1995).
Polymer Phase System Using the Cross-axis Synchronous Coil Planet Centrifuge, J. Chromatogr., 596(1), 118122 (1992). 123. Y. Shibusawa, M. Magiyama, U. Matsumoto, Y. Ito, Complementary use of Counter-current Chromatography and Hydroxyapatite Chromatography for the Separation of Three Main Classes of Lipoproteins from Human Serum, J. Chromatogr., B, 664(2), 295301 (1995). 124. T. Tadey, W.C. Purdy, Effect of Detergents on the Electrophoretic Behaviour of Plasma Apolipoproteins in Capillary Electrophoresis, J. Chromatogr., A, 652(1), 131138 (1993). 125. G. Schmitz, U. Borgmann, G. Assman, Analytical Capillary Isotachophoresis: a Routine Technique for the Analysis of Lipoproteins and Lipoprotein Subfractions in Whole Serum, J. Chromatogr., 320(1), 253262 (1985). 126. R. Bassi, J. Marquardt, J. Lavernie, Biochemical and Functional Properties of Photosystem II in a Agranal
127. G. Schmitz, C. Mollers, Analysis of Lipoproteins with Analytical Capillary Isotachophoresis, Electrophoresis, 15(1), 3139 (1994). 128. L. Zolla, M. Bianchetti, A.M. Timperio, G. Scarascia Mugnozza, D. Corradini, Capillary Electrophoresis of Closely Related Intrinsic Membrane Proteins of the Photosystem II Light-harvesting Complex (LHC II), Electrophoresis, 17, 15971601 (1996). 129. R.M. Garavito, D. Picot, P.J. Loll, Strategies for Crystallizing Membrane Proteins, J. Bioenerg. Biomembr., 28(1), 1327 (1996). 130. T. Barros, A. Royant, J. Standfuss, A. Dreuw, W. Kuhlbrandt, Crystal Structure of Plant Light-harvesting Complex Shows the Active, Energy-transmitting State, EMBO J., 28(3), 298306 (2009).

2011 - Chromatography of Membrane Proteins and Ins

Uploaded by

Document Information

Original Description:

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

2011 - Chromatography of Membrane Proteins and Ins

Uploaded by

Copyright:

Available Formats

Chromatography of Membrane Proteins and Lipoproteins

Lello Zolla and Angelo DAlessandro University of Tuscia, Viterbo, Italy

PEPTIDES AND PROTEINS

CHROMATOGRAPHY OF MEMBRANE PROTEINS

CHROMATOGRAPHY OF MEMBRANE PROTEINS AND LIPOPROTEINS

PEPTIDES AND PROTEINS

CHROMATOGRAPHY OF MEMBRANE PROTEINS AND LIPOPROTEINS

PEPTIDES AND PROTEINS

CHROMATOGRAPHY OF MEMBRANE PROTEINS AND LIPOPROTEINS

PEPTIDES AND PROTEINS

CHROMATOGRAPHY OF MEMBRANE PROTEINS AND LIPOPROTEINS

ELECTROPHORESIS OF MEMBRANE PROTEINS: A BRIEF OVERVIEW

PEPTIDES AND PROTEINS

CHROMATOGRAPHY OF MEMBRANE PROTEINS AND LIPOPROTEINS

PEPTIDES AND PROTEINS

CHROMATOGRAPHY OF MEMBRANE PROTEINS AND LIPOPROTEINS

0.12 0 5 10 15 20 Time (min) 25 30 35 40

Figure 3 Separation of erythrocyte membrane proteins

PEPTIDES AND PROTEINS

H Fd M G e- E L I DF F C H J K B A Lhca 1-4 P700 e N PC F PC Fx A A Lhca 1-4

b6 FeS IV Q cyt b cyt b e QH e FeS cytf

CHROMATOGRAPHY OF MEMBRANE PROTEINS AND LIPOPROTEINS

Freezing at 80 C and homogenization Centrifugation at 1400g for 10 min

Centrifugation at 10 000g for 10 min

Ultracentrifugation at 40 000g for 30 min

Supernatant PSI + PSII

41 000 rpm for 24 h

PEPTIDES AND PROTEINS

CHROMATOGRAPHY OF MEMBRANE PROTEINS AND LIPOPROTEINS

0.02 0 (c) 10 20 Time (min) 30 40

PEPTIDES AND PROTEINS

CHROMATOGRAPHY OF MEMBRANE PROTEINS AND LIPOPROTEINS

45 40 35 30 25 20 15 10 5 0 20 40 Time (min) 40 35 30 25 20 15 10 5 0 20 40 Time (min) 120 60 80 60 80

100 80 60 40 20 0 PsaE PsaH.C 20 Lhca 1 3

Lhca4 Lhca2 PsaA+B PsaF PsaL,F,G

PsaD (c) 40 Time (min) 60 80

PEPTIDES AND PROTEINS

CHROMATOGRAPHY OF MEMBRANE PROTEINS AND LIPOPROTEINS

lhca 3 psa F lhca 2.1 lhca 2.2

psa H psa C psa E

lhca 1.1 lhca 1.2

0 (b) 0 10 20 Time (min) 30 40

PEPTIDES AND PROTEINS

0 0 (c) 10 20 Time (min) 30 40

Signal intensity (counts 108)

Signal intensity (counts 108)

Signal intensity (counts 108)

psb B psb H psb I psb K psb A psb C psb D

CHROMATOGRAPHY OF MEMBRANE PROTEINS AND LIPOPROTEINS

PEPTIDES AND PROTEINS

CHROMATOGRAPHY OF MEMBRANE PROTEINS AND LIPOPROTEINS

PEPTIDES AND PROTEINS

C's A-I 15 Time (min) 20

CHROMATOGRAPHY OF MEMBRANE PROTEINS AND LIPOPROTEINS

Plant species: P, petunia; M, maize; T, tomato; B, barley.

PEPTIDES AND PROTEINS

CHROMATOGRAPHY OF MEMBRANE PROTEINS AND LIPOPROTEINS

COMPARISON WITH OTHER METHODS

PEPTIDES AND PROTEINS

CHROMATOGRAPHY OF MEMBRANE PROTEINS AND LIPOPROTEINS

PEPTIDES AND PROTEINS

CHD CID CN CN-PAGE CPG CTAB-PAGE