Principle of Protein Identification by Tandem MS

Protein tandem mass spectrometry (MS/MS) is a highly precise and sensitive technique widely used in proteomics research. The principle involves using a mass spectrometer to measure the mass-to-charge ratio (m/z) of peptides, which allows for the inference of the protein's sequence and structure. The following section details the principles of protein tandem mass spectrometry identification.

 

Sample Preparation

Protein samples are first subjected to enzymatic digestion, typically using trypsin, to break down the proteins into smaller peptides. These peptides, due to their mass range, are more suitable for mass spectrometry analysis and fall within the mass spectrometer's measurement capabilities.

 

One-Dimensional Mass Spectrometry (MS1)

In one-dimensional mass spectrometry analysis, peptides are ionized through techniques such as electrospray ionization (ESI) or matrix-assisted laser desorption/ionization (MALDI). The ionized peptides enter the mass analyzer of the mass spectrometer, where their m/z ratios are measured, resulting in a mass spectrum of the peptide mixture.

 

Peptide Selection and Fragmentation

In tandem mass spectrometry (MS/MS), specific peptides are selected from the one-dimensional mass spectrum for further analysis. These selected peptides, referred to as precursor ions, undergo fragmentation through collision-induced dissociation (CID) or other fragmentation techniques. This process breaks the precursor ions into smaller fragment ions, which provide partial sequence information of the peptides.

 

Two-Dimensional Mass Spectrometry (MS2)

The fragment ions are analyzed in the second mass analyzer, where their m/z ratios are measured, producing a fragment ion spectrum. By analyzing the masses of these fragment ions, the sequence of the precursor peptide can be deduced. Different fragment types, such as b-ions and y-ions, give insights into specific bond cleavages within the peptide.

 

Data Analysis

The mass spectrometry data obtained are interpreted using database searches and algorithm matching. Databases such as UniProt or NCBI's protein sequence database are commonly used. By matching experimental data with theoretical mass spectra, the sequences of peptides and proteins can be identified. Advanced data processing and analysis algorithms, such as SEQUEST and Mascot, significantly enhance the accuracy and speed of identification.