DE NOVO SEQUENCING OF PROTIEN BY LC-MS/MS ANALYSIS

BY
S.SASI MOUNIKA,
K.V.S.R.SIDDARTHA COLLEGE OF PHARMACEUTICAL SCIENCES,

VIJAYAWADA.

Edman Degradation and MS-MS Analysis

Introduction to Edman Degradation Chemistry of Edman Degradation Applications Introduction to MS-MS spectrometry Principle Instrumentation Bioinformatics for the Identification of proteins based on MS-MS analysis Protein Identification based on peptide masses and MSMS spectra

Edman Degradation
In 1967, a technique called protein sequenator was introduced by Edman and Begg. This technique was used for the N-terminal sequencing of proteins. Edman degradation technique allowed the extraction of individual amino acids in a cycle dependent manner from the Ntermini of proteins.

Chemistry of Edman Degradation

Three steps cycle. 1.Coupling:The technique utilizes Phenylisithiocyanate to react with the N-terminal residue under alkaline condition. 2.Cleavage:The resultant phenylthiocarbamyl (PTC) derivatized amino acid is hydrolyzed in anhydrous acid Trifluoroacetic acid (TFA) so as to release an anilinothiazolinone (AZT) derivative of the amino acid residue. 3.Conversion:The hydrolysis reaction results in a rearrangement of the released N-terminal residue to a Phenylthiohydantoin (PTH) derivative, which is then eluted from an HPLC column.

The retention time associated with the PTH derivative obtained from each repetition of the cycle allows the operator to identify the amino acid sequence of the peptide or protein. By repeating the chemical degradation cycle, it was possible to obtain the amino acid sequence at the N-terminus of typically up to 20 amino acids.

Applications and Limitations

Applications: This approach is widely used to perform the De novo sequencing of a protein. To sequence a sufficient length of the protein to be able to clone the gene. Limitation: It will not work if the N-terminal amino acid has been chemically modified or if it is concealed within the body of the protein. It requires the use of either guess or a separate procedure to determine the positions of disulfide bridges.

MS-MS Spectrometry
Also known as Tandem mass spectrometry It is used to produce structural information about a compound by fragmenting specific sample ions inside the mass spectrometer and identifying the resulting fragment ions. This information can then be pieced together to generate structural information regarding the intact molecule. It also enables specific compounds to be detected in complex mixtures though their characteristic fragmentation patterns may be different.

Principle
The fragmentation is achieved by introducing ion-molecule collisions by a process known as collision-induced dissociation (CID) or Collision activated dissociation. CID is accomplished by selecting an ion of interest with a mass filter/Analyzer and introducing that ion into a collision cell. A collision gas (Ar) is introduced into a collision cell, where the selected ion collides with the argon atoms, resulting in fragmentation. The fragments can then be analyzed to obtain a daughter ion spectrum, which is finally used to obtain structural information.

Instrumentation
A tandem mass spectrometer is a spectrometer that has more than one analyzer, in practice usually there are two. The two analyzer are separated by a collision cell into which an inert gas such as Argon, Xenon is admitted to collide with the selected sample ions and bring about their combinations. The analyzers used are being as Quadrupole quardupole Magnetic sector-quardupole Magnetic sector- Magnetic sector Quadrupole- Time of Flight

MS-MS with a Triple Quadrupole Mass spectrometer

The first mass analyzer utilized for proteomic studies. consists of four rods placed at equidistance as if they were placed on the surface of a cylinder. For triple quadrupole mass analyzer, tandem mass spectrometry utilizes the second quadrupole as a collision cell to generate fragment ions. CID is initiated by selecting an ion [(peptide+H+)] having a particular m/z with the first quadrupole, Q1 Subsequently, only the second quadrupole[Q2, collision cell,] where it collides with argon atoms and fragments. These fragments can then be analyzed with the third quadrupole [Q3] and structural information can be obtained. Technique is used extensively in peptide and carbohydrate sequencing.

MS-MS with a Two Sector Mass Spectrometer (Linked Scaning)

Two sector or double focusing mass spectrometers were developed to perform accurate mass measurement. Could be scanned in special ways so that metastable decomposition could be observed without inference from source generated ions. This was done by linking the magnetic and electrostatic fields such that they were scanned together. This results in obtaining reasonable product ion spectra. These instruments are not considered tandem mass spectrometers, yet they merit mention because of the tandem-like information the can give.

MS-MS with a Four Sector Mass Spectrometer (Magnet ESA-Magnet ESA)

Here, the ions can be selected out by the first two sectors, followed by mass analysis in the second two sectors. As with the triple quadrupole, structural information can be obtained. Application: Used in peptide sequencing. Advantage: High collisions cause significant fragmentations and facilitate the acquisition of detailed structural information. Disadvantage: High cost and size.

MS-MS with a Two-sector (Magnetic ESA) Quadrupole Mass Spectrometer

MS-MS with the two sector magnetic electrostatic Mass Analyzer is also common. But these hybrid instruments are not so accurate as the sour sector magnetic/electrostatic instruments.

Bioinformatics for the Identification of proteins based on MS-MS analysis

All the ESI based tandem mass spectrometers are generally used to perform MS and MS-MS spectra. Contain information that can be used to identify a peptide provenance by proteins/DNA database searching though the mass accuracy, resolution, sensitivity and fragmentation patterns are different. The MS-MS spectra contain the fragmentation patterns related to the amino acid sequence of specific peptides. The approaches used for the interpretation of these spectra are classified into three subgroup. No interpretation Partial Interpretation De novo sequencing

De novo Sequencing
The full interpretation of the MS/MS spectra is called the de novo sequencing. The MS/MS spectra of peptide contains ladder type information which in principle, indicates their amino acid sequence.

Protein Identification based on Peptide Masses and MS/MS Spectra

The MS/MS spectra contain the fragmentation pattern related to the amino acid sequence of specific peptides. Three approaches are used for the interpretation of MS/MS spectra which can be classified according to the level of user intervention required. In the first subgroup- the information contained in the spectra is directly correlated with protein/DNA sequence information contained in databases. In second subgroup, partial interpretation of the MS/MS spectra is involved and therefore require human intervention. In last subgroup, often called DE NOVO SEQUENCING of proteins, is used as a last resource when no matching information is available in databases and the quality of the MS/MS spectra is good. The MS/MS spectra of peptide contain ladder-type information which in principle, indicates their amino acid sequences.

Oligonucleotide sequencing by Tandem Mass/ MS/MS spectrometry

Oligonucleotide sequencing is achieved by MS/MS spectrometry though it is not so well documented. Experimental principle, similar to that of peptide sequencing, in which individual species are mass measured in MS mode of instrument operation, then their molecular-related ions selected by the first (Quadrupole) analyzer to be transmitted into the collision cell where they undergo fragmentation after bombardment with a collision gas. The fragments are analyzed by the second (TOF) analyzer to produce an MS-MS product, or daughter ion spectrum showing all the fragment ions that arise directly from the chosen parent or precursor ions.

Multidimensional Liquid Chromatography Tandem Mass Spectrometry

Often more complex samples (such as serum) will require additional fractionation steps in order to identify proteins of lower abundance. In practice, LC-MS/MS methods have a dynamic range of roughly 3 orders of magnitude, while the detection limit of the mass spectrometer may be femtomolar or less, the presence of peptides at high concentrations will mask the presence of lower abundance ions. In order to extend the dynamic range of measurement, additional chromatographic or affinity steps have been developed to simplify mixtures as a part of the identification strategy.

ACKNOWLEDGEMENT
G.DEVALARAO
N.KANAKA DURGA DEVI AZIZ UNISA
KVSR SIDDARTHA COLLEGE OF PHARMACEUTICAL SCIENCES, VIJAYAWADA.