Workflow of Protein Sequencing Using Mass Spectrometry

Protein mass spectrometry (MS) is a powerful tool used for the identification and quantification of proteins. It plays a critical role in proteomics research by separating, detecting, and identifying proteins through mass spectrometry technology.

 

Sample Preparation

Sample preparation is the first step in protein mass spectrometry sequencing. Ensuring the purity and stability of the sample is crucial. Sample preparation typically includes the following steps:

 

1. Protein Extraction

Extract proteins from cell or tissue samples.

 

2. Protein Digestion

Use enzymatic digestion (e.g., trypsin digestion) to cleave large protein molecules into smaller peptides.

 

3. Sample Purification

Remove impurities from the sample using various chromatographic techniques (e.g., reverse-phase high-performance liquid chromatography, RP-HPLC).

 

Sample Separation

Before mass spectrometry analysis, the sample needs further separation. Liquid chromatography (LC) is commonly used to separate complex peptide mixtures into individual components. Common separation techniques include:

 

1. High-Performance Liquid Chromatography (HPLC)

Separate peptides based on their hydrophilicity and hydrophobicity.

 

2. Multidimensional Liquid Chromatography (MudPIT)

Combine strong cation exchange (SCX) and reverse-phase liquid chromatography (RP-LC) for multidimensional separation of complex samples.

 

Mass Spectrometry Analysis

Mass spectrometry analysis is the core step of protein mass spectrometry sequencing, detecting and identifying peptides through a mass spectrometer. It mainly includes the following steps:

 

1. Electrospray Ionization (ESI) or Matrix-Assisted Laser Desorption/Ionization (MALDI)

Ionize peptides into gas-phase ions.

 

2. Detection by Mass Spectrometer

Common mass spectrometers include triple quadrupole (QqQ), time-of-flight (TOF), and orbitrap, which separate and detect ions based on the mass-to-charge ratio (m/z).

 

3. Tandem Mass Spectrometry (MS/MS)

Further fragment ions through two or more stages of mass spectrometry (MS^n) to provide more detailed structural information.

 

Data Analysis

Data analysis of mass spectrometry is crucial for protein identification and quantification. Data analysis typically includes the following steps:

 

1. Preprocessing of Mass Spectrometry Data

Including peak detection, denoising, and alignment.

 

2. Peptide Matching

Use database search algorithms (e.g., Mascot, Sequest) to match experimental mass spectrometry data with theoretical mass spectrometry data.

 

3. Protein Identification

Reconstruct protein sequences based on peptide matching results.

 

4. Quantitative Analysis

Perform relative or absolute quantification of proteins using labeling methods (e.g., iTRAQ, TMT) or label-free methods (e.g., LFQ).

 

Results Validation

The results of mass spectrometry analysis need to be validated by other methods to ensure their accuracy and reliability. Common validation methods include:

 

1. Western Blotting

Validate the presence and expression levels of specific proteins.

 

2. Quantitative PCR (qPCR)

Validate the gene expression levels related to the proteins.

 

3. Functional Assays

Validate the biological functions of proteins through biological assays (e.g., cell proliferation, apoptosis analysis).

 

MtoZ Biolabs provides integrate protein sequencing service by mass spectrometry.