Workflow of Peptide Sequencing

Peptide sequencing is an essential method for studying the structure and function of proteins. By determining the sequence of amino acids in a peptide chain, it aids scientists in understanding the biological functions of proteins and their roles within organisms. The primary steps of peptide sequencing include sample preparation, peptide generation, peptide separation, mass spectrometry analysis, data processing, and result interpretation.

 

Sample Preparation

Sample preparation is the first step in peptide sequencing, involving the extraction of proteins from biological samples. The main objective is to obtain pure proteins for subsequent steps. Common methods include cell lysis, ultracentrifugation, and protein purification. The quality of sample preparation significantly influences the accuracy and efficiency of peptide sequencing.

 

Peptide Generation

Once pure protein samples are obtained, they need to be cleaved into smaller peptides. This step is typically achieved through protease digestion, with trypsin being the most commonly used protease due to its specificity in cleaving peptide bonds after lysine and arginine residues. Besides enzymatic digestion, chemical methods such as cyanogen bromide (CNBr) cleavage at methionine residues can also be used.

 

Peptide Separation

The resulting peptide mixture needs to be separated for subsequent mass spectrometry analysis. Common separation methods include liquid chromatography (LC) and capillary electrophoresis (CE). High-performance liquid chromatography (HPLC) is widely utilized in peptide separation, enabling the separation of complex peptide mixtures based on their physicochemical properties, such as hydrophilicity and hydrophobicity.

 

Mass Spectrometry Analysis

Mass spectrometry (MS) is the core technique in peptide sequencing. Mass spectrometers ionize peptides and measure their mass-to-charge ratios (m/z) to determine their mass. Common mass spectrometers include electrospray ionization mass spectrometry (ESI-MS) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Mass spectrometry typically involves both MS and MS/MS stages, where the initial MS determines the molecular weight of peptides, and the subsequent MS/MS induces peptide fragmentation to obtain amino acid sequence information.

 

Data Processing

Data generated from mass spectrometry require sophisticated processing to derive the amino acid sequence of peptides. Data processing software matches the peak information from mass spectra with known protein sequences using database search algorithms. Common software includes Mascot, Sequest, and Andromeda.

 

Result Interpretation

The final step is to translate peptide sequencing data into biological information. Scientists compare the sequencing results with known protein sequences in databases to identify protein types and functions. Additionally, result interpretation includes analyzing peptide modifications (e.g., phosphorylation, acetylation) and predicting protein three-dimensional structures.