Peptide Sequence Determination by MS-MS

Peptide sequence determination is a core aspect of proteomics research, involving the precise identification of the sequences of proteins or peptides. MS-MS, or tandem mass spectrometry, is a powerful technique for achieving this. This article will introduce the basic principles of peptide sequence determination, the working mechanism of MS-MS technology, and its applications in biomedical research.

 

The sequence determination of proteins or peptides is based on the identification of their constituent amino acids. Each protein is a long chain molecule made up of 20 types of amino acids in different orders, this order is known as the protein's sequence. To determine a protein's sequence, in essence, is to determine the arrangement order of these amino acids.

 

MS-MS Technology

Tandem mass spectrometry achieves peptide sequence determination through two-stage mass spectrometry analysis. Firstly, in the first stage mass spectrometry (MS1), mixed protein samples are ionized into charged molecules (ions), and then separated by mass-to-charge ratio (m/z). Then, specific ions are selected for the second stage mass spectrometry (MS2) analysis. In MS2, these selected ions are further broken down into smaller fragment ions. By analyzing the mass-to-charge ratios of these fragment ions, the amino acid sequence of the original peptide can be inferred.

 

Process Steps

1. Sample Preparation

(1) Protein Extraction

Proteins are extracted from biological samples and cut into peptides by enzymes (such as trypsin).

 

(2) Peptide Purification

Cut peptides are purified using chromatography techniques (such as liquid chromatography) to reduce sample complexity and improve the sensitivity of subsequent analysis.

 

2. MS/MS Analysis

(1) First Stage Mass Spectrometry (MS1)

Peptide samples are introduced into the mass spectrometer and ionized (usually using electrospray ionization, ESI, or matrix-assisted laser desorption/ionization, MALDI). The mass-to-charge ratios (m/z) of the ionized peptide ions are measured in the first stage mass spectrometry.

 

(2) Ion Selection and Fragmentation

Specific peptide ions are selected based on their m/z values and sent to the collision cell, where they are fragmented. This process generates a series of fragment ions representing different parts of the original peptide sequence.

 

(3) Second Stage Mass Spectrometry (MS2)

The mass-to-charge ratios of these fragment ions are measured in the second stage mass spectrometry, generating a spectrum, i.e., the MS/MS spectrum.

 

Sequence Determination Spectrogram Analysis

MS/MS profiles were analyzed to identify the type and order of fragment ions, and this information was used to infer the amino acid sequence of the original polypeptide. Database Search: MS/MS data were matched to a known protein database using specific software (such as Mascot, SEQUEST, or MaxQuant) to determine the sequence of polypeptides.

 

MS-MS technology is a powerful tool, crucial for understanding the molecular mechanisms of biological systems. By precisely identifying the sequences of proteins and peptides, it has helped scientists unravel many secrets of life processes, advancing biomedical research. With the advancement of technology and the expansion of applications, MS-MS will continue to play an important role in proteomics and related fields.