Methods 35 (2005) 248–255

www.elsevier.com/locate/ymeth

Protein identiWcation using 2D-LC-MS/MS

Claire Delahunty, John R. Yates III¤
Department of Cell Biology, The Scripps Research Institute, La Jolla, CA 92037, USA

Accepted 25 August 2004

Available online 12 January 2005

Abstract

Multidimensional liquid chromatography techniques have been coupled to tandem mass spectrometry to provide a robust
method to identify proteins in complex mixtures. Data acquisition is interfaced directly with search algorithms for identiWcation
through cross-correlation with databases. This review describes the most recent advances in methodologies for protein identiWcation
by mass spectrometry and describes the limitations of the application of the technologies.
 2004 Elsevier Inc. All rights reserved.

Keywords: Proteomics; Multidimensional chromatography; Mass spectrometry; Protein mixture analysis

1. Introduction theoretical spectra generated from protein and nucleo-

Mass spectrometry-based methods for the identiWca-
tion of proteins have become a standard platform in
proteomics. The most popular MS-based strategies rely
on proteolytic digestion of proteins into peptides before
introduction into the mass spectrometer. Digestion of
proteins into similar sized peptides helps to overcome
the solubility and handling problems associated with
proteins and creates peptide fragments which are easily
ionized in the mass spectrometer. Peptide ions are Wrst
measured as intact fragment ions, then selected based on
their m/z and subject to collisionally induced dissocia-
tion (CID) [1,2], in a process known as tandem mass
spectrometry (MS/MS). Under the low-energy condi-
tions employed for CID, peptide ions fragment in pre-
dictable patterns. Because fragmentation patterns are
predictable, theoretical spectra can be constructed for
sequences in protein or nucleotide databases. Computer
algorithms use the CID fragmentation patterns of sam-
ple peptides to determine the sequence of the peptide,
and this sequence information is used to search against
*
Corresponding author. Fax: +1 858 784 8883.
E-mail address: jyates@scripps.edu (J.R. Yates III).
tide databases. Protein identiWcations are made by Wnd-
ing the best correlation between experimentally derived
sequence information and sequences in the database.
One of the greatest strengths of tandem mass spec-
trometry for protein identiWcation is the inherent ability
to sequence peptides directly from mixtures. Thus, mass
spectrometry allows the direct identiWcation of the indi-
vidual constituents of protein complexes involved in a
wide range of physiological functions. However, if the
mixture of peptides is highly complex, it is advantageous
to use a separation step prior to analysis to limit the
number of peptides the mass spectrometer sees over the
time of the analysis. The method most commonly used
to reduce sample complexity prior to introduction into
the mass spectrometer is the separation of sample pro-
teins by gel electrophoresis followed by excision of the
individual protein spots from the gel and in-gel digestion
with a protease (i.e., trypsin). One-dimensional (1D) gels,
which separate proteins based on molecular mass,
provide a low-resolution separation of proteins, but
when coupled with tandem mass spectrometry can be
used to identify proteins in moderately complex mix-
tures. For more complex mixtures which are not suY-
ciently resolved in a 1D separation, a multidimensional

1046-2023/$ - see front matter  2004 Elsevier Inc. All rights reserved.
doi:10.1016/j.ymeth.2004.08.016
 C. Delahunty, J.R. Yates III / Methods 35 (2005) 248–255
separation may be necessary. Multidimensional separa-
tions exploit two or more independent physical proper-
ties of the proteins or peptides to achieve a higher level
of resolution and higher loading capacity than can be
achieved in a single dimension. Separation strategies can
be selected so that components not separated in the Wrst
dimension are separated in the second. Two-dimensional
(2D) gels are the most common multidimensional sepa-
ration technique used to separate proteins in complex
mixtures. In this technique, proteins are separated on the
basis of their iso-electric point in the Wrst dimension and
by their molecular mass in the second dimension. The
position occupied by a protein on a 2D gel is a reXection
of its approximate pI and mass. Although gel-based sep-
arations of proteins prior to analysis are eVective meth-
ods for the analysis of a large number of proteins in
complex mixtures, the methods have many shortcom-
ings. One-dimensional gels provide a separation method
that is labor intensive, aVords limited resolution of pro-
teins, has limited dynamic range, and decreases the
detection sensitivity in the mass spectrometer. To visual-
ize gel-puriWed proteins so they can be excised and
extracted from the gel, the gels must be stained, either by
silver staining, Coomassie staining or staining with Xuo-
rescent dyes. (Staining gels also allows a quantitative
comparison of protein expression, albeit in a limited
dynamic range.) Two-dimensional gels provide better
resolution of proteins but are still labor intensive and
have a limited dynamic range. In addition, 2D gels are
unable to detect membrane, highly basic or highly acidic
proteins under standard conditions.
Gel-based proteomics strategies are rapidly being
supplanted by methods that involve peptide separation
via high eYciency nanocolumn liquid chromatography
separation techniques directly linked to a mass spec-
trometer. In these methods, complex protein mixtures
are reduced to peptides prior to separation and the total
peptide mixture is loaded onto a nanocolumn. The nano-
column is made from fused silica microcapillary tubing
that is typically 50–100
m in diameter and has a tip that
has been pulled to an inner diameter of 2–5
m [3–6]. For
single dimensional separations, the nanocolumn is Wrst
packed to 10 cm with reverse-phase C18 material. Once
loaded onto the nanocolumn, peptides are eluted directly
into the ionization source of the mass spectrometer using
an HPLC acetonitrile gradient. The gradient is run at
very low Xow rates, typically 100–200 nL/min and the
typical elution time for peptides is 10–30 s. Stable elec-
trospray at the front of the mass spectrometer’s heated
capillary is produced when a voltage of 1.8–2.4 kV is
applied to the precolumn liquid–metal interface. As pep-
tides enter the mass spectrometer, a survey scan of the
intact peptides is obtained. Using data-dependent acqui-
sition, the instrument can be set to automatically moni-
tor the survey scan and select peptides based on pre-set
criteria such as intensity, charge state or m/z. The
249
selected peptides are fragmented using CID, and MS/MS
spectra are collected. By coupling an LC system with a
tandem mass spectrometer and data-dependent scan-
ning, it is possible to distinguish individual proteins in
complex mixtures containing more than 50 components
without prior puriWcation [7–9].
While LC-MS/MS is routinely used to sequence
peptides and identify proteins directly from complex
mixtures, some samples present complexity beyond the
separation capacity of a 1D LC technique. To achieve
enhanced separation, gel electrophoresis can be
employed to separate intact proteins prior to digestion
and loading on the nanocolumn. However, this
approach is still encumbered by the shortcomings inher-
ent in gel-based techniques. Recently, a higher-resolu-
tion and higher-capacity 2D separation has been
achieved with an in-line system using a biphasic
nanocolumn [10]. In this technique, known as multidi-
mensional protein identiWcation technology (MudPIT),
a 3–5 cm section of strong cation exchange resin (SCX)
is placed directly upstream from the C18 resin in the
nanocolumn. Because the SCX segment has a much
greater loading capacity than the RP segment, it acts as a
peptide reservoir, storing peptides until a subset is
“bumped” to the RP segment with incremental increases
in salt in the LC gradient. The dislodged peptides are
separated on the RP phase using an acetonitrile gradient
and, after re-equilibration, another fraction of peptides
is displaced from the SCX to the RP with an increase in
salt concentration. The iterative process of salt bump
followed by RP separation is repeated until the reserve
of peptides on the SCX is exhausted. This method
greatly increases the number of digested proteins that
can be analyzed and enhances the detection of low-
abundance proteins in the mixture. The power of 2D-
LC-MS/MS for protein identiWcation has been shown in
the analysis of total cell lysate of Saccharomyces cerevi-
siae [11]. This study established that the MudPIT is
unbiased, since large or small proteins of high or low-
abundance, or extremes in pI were identiWed with equal
sensitivity. Additionally, it was the Wrst LC-MS/MS
study to identify a signiWcant number of membrane pro-
teins. MudPIT has been used to idenitify proteins in
samples from a wide variety of sources [12–15] and has
been successfully applied to the identiWcation of post-
translational modiWcations [16], as well as the quantita-
tive comparison of protein expression using stable
isotope labeling [17,18].
Two-dimensional separation prior to MS/MS has
also been performed by coupling an oV-line SCX sepa-
ration with either an oV-line or on-line RP separation
prior to introduction to the mass spectrometer [19,20].
In this approach, fractions are collected after the
sample is run on an SCX column and each fraction is
reduced in volume and run on an LC-MS/MS reverse-
phase nanocolumn. One advantage of an oV-line
250 C. Delahunty, J.R. Yates III / Methods 35 (2005) 248–255
separation over in-line techniques is that it allows sam-
ple manipulation and separation optimization between
dimensions. Because each separation phase is indepen-
dent, there is more Xexibility in choices for the compo-
sition of the columns, the buVers, and the length of the
gradient. However, the independent optimization of
each phase of separation can result in prohibitively
long run times.
Two-dimensional LC-MS/MS methods have been
shown to be useful for many applications, but complex
mixtures of peptides frequently contain salts which can
interfere with the interaction of the peptides with the
SCX resin. For these samples, oV-line desalting can be
carried out prior to MudPIT using a solid phase extrac-
tion column. Alternatively, desalting can be performed
online using a triphasic column which contains a 3 cm
segment of C18 packing material directly upstream from
the SCX segment. In this technique, peptides are
desalted in the Wrst cycle and advanced to the SCX seg-
ment where they are subject to multidimensional separa-
tion [21].
Optimization of MudPIT is dependent on sample
concentration, since the sensitivity of peptide detection
in the mass spectrometer is determined by the concentra-
tion of the peptide eluted from the column. To optimize
detection of the lowest abundance peptides, it is typical
to heavily load the column, create small increments in
the salt “bumps” to displace peptides from the SCX, and
run a long RPLC gradient. In this method, it is common
to see highly abundant peptides elute over a number of
diVerent salt concentrations. Under most circumstances,
this will not interfere with the elution and identiWcation
of lower abundant peptides. However, with limited sam-
ple quantity longer gradients should be avoided since
they may actually decrease detection sensitivity for low-
abundance peptides. When sample quantity is limited it
is often useful to optimize conditions using a standard
protein mixture of similar concentration. With a well-
optimized separation it is now routine to identify 1500–
2000 proteins from a sample derived from a whole cell
lysate.
The success of protein mixture analysis by LC-LC/
MS-MS depends on the chromatographic step used to
introduce the sample to the mass spectrometer. To
achieve good chromatography, high quality nanocol-
umns are necessary. A successfully packed nanocolumn
will allow the Xow rate (200–300 nL/min) required for
femtomole sensitivity. If solvent Xow is not seen during
the packing step, attempts can be made to gently score
the tip with a ceramic scribe. If the column clogs during
sample loading, it is frequently a sign that the sample has
not been suYciently centrifuged prior to loading. Occa-
sionally, salts in the sample can interfere with loading
and in these situations the clog can be cleared by brieXy
immersing the column tip in boiling water. It is possible
to use nanocolumns more than once, but extreme care
must be taken to make sure all previous sample has
been stripped from the column prior to re-loading. Col-
umns must be re-equilibrated after stripping and special
care must be taken to make sure the solvent Xow is
satisfactory.
MudPIT analysis of complex mixtures of proteins is
most often constrained by the amount of data generated
in each experiment. Currently, a single MudPIT analysis
can be run in 6–24 h (with an additional 2–7 h for col-
umn packing and sample loading) and result in over
70,000 spectra [22]. The Wrst step in analysis of the col-
lected data is the comparison of experimental MS/MS
spectrum against theoretical spectrum generated from
database sequence information. This step can take any-
where from a few hours to a few days, depending on the
size of the database being searched, the complexity of
the sample, and the computing resources available. Once
peptide “hits” are scored for the identiWcation of pro-
teins, manual validation to verify real protein identiWca-
tions versus false positives is frequently necessary. While
this process has been streamlined by the development of
computer programs which remove spectra of poor qual-
ity, parallelize searches over many computers, Wlter and
sort data, normalize cross-correlations scores reported
by SEQUEST, and aid in assembly [23,24,16], interpreta-
tion of proteomic data remains a possible bottleneck in
the identiWcation of proteins using the MudPIT
technique.
2. Description of method
2.1. Materials
1. Instruments and equipment
LCQ tandem mass spectrometer (Finnigan MAT, San
Jose, California) or other mass spectrometer capable
of tandem mass spectrometry with automated data
acquisition.
Multidimensional HPLC system (Integral Microana-
lytical System, Perseptive Biosystems, Framingham,
MA) or other system capable of multidimensional
chromatography.
Stainless-steel pneumatic pressure bomb (The Scripps
Research Institute, La Jolla, CA or Cytopea).
CO2 laser puller (P250, Sutter Instruments, Novato,
CA) or commercially prepared nanocolumns (New
Objective, Woburn, MA).
PEEK Microcross, Microtight tubing sleeves
(Upchurch ScientiWc).
100 £ 365
m fused silica capillary tubing (Polymicro
Technologies, Phoenix, AZ).
Aqua C18 reverse-phase 5
m resin (Phenomenex,
Torrance, CA).
Partisphere strong cation exchange (SCX) resin
(Whatman, Clifton, NJ).
 C. Delahunty, J.R. Yates III / Methods 35 (2005) 248–255
2. Chemicals and enzymes
Cyanogen bromide, formic acid, urea, ammonium
acetate, iodoacetamide, trichloroacetic acid, Trizma
HCl (Sigma).
HPLC-grade acetonitrile, HPLC-grade methanol
(Fisher ScientiWc, Fairlawn, NJ).
Tris(2-carboxyethyl)phosphine (TCEP) (Pierce,
Rockford, IL).
Ammonium bicarbonate (J.T. Baker, Phillipsburg,
NJ).
Acetic acid, calcium chloride (Mallinckrodt Baker,
Paris, KY).
Sequencing-grade trypsin (Promega, Madison, WI).
Sequencing-grade endoproteinase Lys-C (Roche
Diagnostics, Indianapolis, IN).
2.2. Methods
1. Sample preparation for MudPIT analysis
A. Sample pre-treatment
(a) Centrifuge at 4000 rpm for 30 min to separate
the soluble from insoluble fractions.
(b) For soluble fractions, skip to B. “Reduction
and Carboxyamidation.”
(c) For insoluble fractions, add 50–100
L of a
solution of CNBr (500 mg/mL in 96% formic
acid) and incubate at RT, in the dark, over-
night. Neutralize with ice-chilled 30% ammo-
nium hydroxide. Carefully adjust to pH 8–8.5
using ammonium bicarbonate.
B. Reduction and carboxyamidation
(a) Precipitate the sample with trichloroacetic acid
in a microcentrifuge tube.
(b) Resuspend the sample in 20
L of 100 mM
Tris, pH 8.5, 8 M urea (more buVer can be used
if necessary).
(c) Add TCEP to a Wnal concentration of 5 mM
and incubate at RT for 15 min.
(d) Add 1 M iodoacetamide to a Wnal concentra-
tion of 25 mM and incubate in the dark at RT
for 20 min.
C. Enzymatic digestion
(a) Add endoproteinase Lys C (1:100 enzyme:sam-
ple).
(b) Incubate with shaking at 37 °C for a minimum
of 8 h in the dark.
(c) Dilute sample to 2 M urea, 50 mM ammonium
bicarbonate, pH 8.5.
(d) Add 100 mM CaCl2 to a Wnal concentration of
1 mM.
(e) Add trypsin (1:100 enzyme:sample).
(f) Incubate with shaking at 37 °C for a minimum
of 8 h in the dark.
(g) Centrifuge sample in a microcentrifuge at max-
imum speed to remove insoluble material and
transfer supernatant to new microcentrifuge
251
tube. Failure to centrifuge well may result in
clogged columns when loading.
2. Column preparation
A. Column pulling
(a) Cut a 50–54 cm length of 100 £ 365
m fused
silica capillary tubing. Hold the center of the
tubing over an alcohol burner and burn the
polyimide coating oV a 5–10 cm section.
Remove the charred material by cleaning with
a Kimwipe soaked in methanol. The tubing
should be clear in this section.
(b) Place the length of tubing in the CO2 P-200
laser puller (Sutter Instruments, Novato, CA)
so that the clear section is in the mirrored
chamber of the puller. In this position, the laser
is focused on the center of the tubing and the
fused silica can be melted.
(c) Select the program on the laser puller that will
result in a 3–5
m i.d. tip. The optimal program
must be experimentally determined, but typical
settings can be found in the manual for the
laser puller.
(d) Alternatively, pre-pulled columns may be
obtained from New Objective (Woburn, MA).
B. Column packing
(a) Place a small amount (t1 mg) of C18 reverse-
phase packing material (5
m) (Polaris C18-A
or similar) in a microcentrifuge tube and add
0.5 mL of methanol. Agitate the tube to create
a slurry of the packing material.
(b) Place the open microcentrifuge tube into a
stainless steel bomb (see Fig. 1) and secure the
lid by tightening the Wve screws.
(c) Insert the Xat end of the pulled nanocolumn
through the ferrule until it reaches the bottom
of the microcentrifuge tube. Tighten the ferrule
until the capillary does not move when gently
tugged.
(d) Adjust the pressure on the helium tank to 400–
800 psi. Slowly pressurize the bomb by opening
the valve on the high-pressure line. If the bomb
is pressurized too rapidly, the microcentrifuge
tube can rupture.
(e) When pressure is applied to the bomb, packing
material should begin to rise and Wll the nano-
column. If packing material stops Wlling the
capillary before 7–10 cm has been packed,
release the pressure on the bomb, loosen the
ferrule holding the capillary, and gently tap the
nanocolumn on the bottom of the microcentri-
fuge tube. Re-tighten the ferrule and re-pres-
surize the bomb. Continue packing until the
desired amount of packing material is loaded.
(f) If a two- or three-phase nanocolumn is to be
made, release the pressure on the bomb and
remove the column. Open the bomb and
252 C. Delahunty, J.R. Yates III / Methods 35 (2005) 248–255
replace the C18 packing material with a micro-
centrifuge tube containing a methanol slurry of
SCX (5
m Partisphere strong cation
exchanger, Whatman) packing material.
(g) Re-pressurize the bomb and load 3 cm of SCX
packing material. For a three-phase column,
repeat the procedure, but load 3 cm of C18
packing material.
C. Setting up the PEEK MicroCross
(a) Attach a 50 £ 365
m section of fused silica
capillary to one of the connection points on the
MicroCross. The other end should be con-
nected to the HPLC pump.
(b) Attach a 20 cm section of microcapillary tub-
ing to the next connection point (clockwise
from the HPLC line) to serve as the split line.
The split line allows very low Xow rates to be
achieved through the nanocolumn. To increase
the Xow rate, a longer split line may be used; to
decrease, a shorter split line is used.
(c) Insert a 1 cm length of gold wire at the third con-
nection point. The gold electrode imparts a volt-
age of approximately 1800 V on the solvent
exiting the needle, allowing electrospray to occur.
(d) The packed nanocloumn is inserted in the
fourth connection point.
(e) The PEEK MicroCross is placed on a stage
(see Fig. 2) which supports the column and the
other connections. The stage has an XYZ
manipulator which allows the needle of the
column to be positioned near the opening of
the mass spectrometer.
D. Sample loading
(a) Pre-equilibrate the nanocolumn with buVer A
(5% acetonitrile, 0.1% formic acid, and 94.9%
H2O) for 5–10 min.
(b) Acidify the sample using 0.5% acetic or 0.5%
formic acid (pH < 3).
(c) Place the sample in the high pressure bomb.
(d) Close the bomb, tighten the lid, and insert the
nanocolumn (needle end up) through the fer-
rule until the bottom of the column touches the
bottom of the microcentrifuge tube.
(e) Begin loading the sample by pressurizing the
bomb. Loading progress can be monitored by
observing the buVer displaced from the column.
(f) Slowly depressurize the bomb to avoid disturb-
ing the packing material. Sample is now loaded
onto the column.
3. Analyzing the sample
A. Multidimensional chromatography
(a) Split the Xow from the HPLC with the PEEK
MicroCross so that the eVective Xow rate from
the HPLC is 0.15–0.25
L/min.
(b) Run a 6-step salt gradient, or a 12-step gradi-
ent for highly complex or concentrated sam-
ples. A 12-step gradient simply requires smaller
increments in the proportion of buVer C
(500 mM ammonium acetate, 5% acetonitrile,
and 0.1% formic acid) used in iterative steps. A
sample six step HPLC gradient is shown in
Table 1.
B. MS/MS Analysis
As peptides elute from the nanocolumn into the
mass spectrometer, scans are acquired in a data-
dependent manner. In this way, the mass spectrom-
eter acquires one conventional scan over the m/z
range 400–1400. Ions detected above a preset ion
current threshold are automatically selected and
subject to tandem mass spectrometry (MS/MS).
Typically, three scans of MS/MS are recorded for
each conventional scan; the Wrst MS/MS scan rep-
resents the most intense ion in the conventional
scan, the second scan represents the second most
intense ion, and the third represents the third most
intense ion. Dynamic exclusion is applied during
the data-dependent acquisition which prevents an
abundant ion from being continually selected for
MS/MS. Once selected, the peptide ion is not
selected again for a period of time so that other,
less intense ions can be analyzed.
C. Database searching
Because peptides fragment in a predictable
manner in a CID process, sequences from dat-
abases can be used to generate theoretical frag-
mentation spectra which can be used to match the
observed MS/MS spectra in the acquired data. A
match of the theoretical MS/MS spectrum of an
amino acid sequence at least seven residues in
length to an experimentally observed peptide MS/
MS spectrum allows identiWcation of the protein
from which the peptide was derived. Several algo-
rithms exist which use correlation between theoret-
ical and experimental fragmentation patterns of
peptides to make identiWcations of proteins,
including SEQUEST [9], which can be run directly
on the LCQ ion trap mass spectrometer. Alterna-
tively, web-based search tools are available at http://
prospector.ucsf. edu, http://www.matrixscience.
com, and http://prowl. rockefeller.edu/PROWL/
pepfagch.html. SEQUEST Wrst looks for all the
amino acid sequences in the database that match
the measured mass of the peptide, and then theo-
retically calculates and matches the expected frag-
ment ion masses against observed MS/MS data.
The best matching peptides are scored using two
separate scoring strategies. Initially, a preliminary
score (Sp) is generated which selects the top 500
peptide sequences and then an independent fast
Fourier transform (FFT) cross-correlation analy-
sis is made to provide a Wnal ranking of the best
matches. A cross-correlation value is reported
 C. Delahunty, J.R. Yates III / Methods 35 (2005) 248–255 253

Fig. 1. Pressure loading device that can be used to pack nanocolumns and to load samples onto nanocolumns.

Fig. 2. Nanocolumn positioned at the opening to the mass spectrometer by a stage and PEEK MicroCross.

which is a measure of the quality of the match
between the sequence and the spectrum. The diVer-
ence between the correlation score for the top-
scoring match and the second-ranked is a measure
of the quality of the match versus other possible
sequences. TurboSEQUEST is a modiWcation of
SEQUEST which has allowed much faster
searches, and other modiWcations have been made
to allow searching for protein modiWcations
[25,26], searching of MALDI-PSD data [27], and
searching of high-energy CID data [28].
Databases to be used in SEQUEST searches can
be obtained from the National Center for Biotech-
nology Information (NCBI) (ftp://ncbi.nlm.nih.gov),
254 C. Delahunty, J.R. Yates III / Methods 35 (2005) 248–255

Table 1 ter, to improvements in the mass accuracy, resolution

Typical 6-step MudPIT HPLC gradient program and speed of the mass spectrometer, and to the develop-
Step Time (min) %A %B %C ment of software to analyze the mass spectrometric data.
1 0 100 0 0 Methods which couple LC/LC directly to the mass spec-
1 5 100 0 0 trometer are rapidly replacing gel-based methods
1 80 55 45 0 because they provide a drastic speed advantage.
1 90 0 100 0
Advances in LC/LC-MS/MS have made possible the
1 100 0 100 0
global identiWcation of proteins from complex mixtures,
2 0 100 0 0 the detection of covalent modiWcations on proteins, and
2 3 100 0 0
2 3.1 95 0 5
the comparative quantitative analysis of proteins.
2 5 95 0 5 Future developments in multidimensional LC-MS/
2 5.1 100 0 0 MS techniques will most likely include the development
2 15 85 15 0 of methods with more than two dimensions which will
2 60 75 25 0 improve the resolution, sensitivity, and dynamic range of
2 112 45 55 0
the technique. Improvements in separation and detec-
3 0 100 0 0 tion will make routine the direct analysis of complex
3 3 100 0 0
protein mixtures of entire proteomes. However, success
3 3.1 87.5 0 12.5
3 5 87.5 0 12.5 in the development of methods to rapidly generate vast
3 5.1 100 0 0 amounts of mass spectral data needs to be accompanied
3 15 85 15 0 by improvements in the speed and accuracy with which
3 60 75 25 0 the data can be analyzed.
3 112 45 55 0
4 0 100 0 0
4 3 100 0 0
4 3.1 75 0 25
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