Workflow of Mass Spectrometry-based Protein Sequencing

Protein sequencing is an essential biotechnological method for determining the amino acid sequence of proteins, crucial for understanding their function, structure, and roles in biological systems. The main steps in protein sequencing methods include sample preparation, protein separation, protein cleavage, peptide separation, peptide identification, and data analysis.

 

1. Sample Preparation

The first step in protein sequencing is preparing the sample, which involves extracting proteins from biological materials using techniques like cell disruption and centrifugation. The extracted protein samples often require purification to eliminate impurities and non-protein substances. Common purification methods include gel filtration, ion exchange, and affinity chromatography.

 

2. Protein Separation

Once extracted and purified, proteins typically need further separation to facilitate subsequent sequencing steps. Key methods for protein separation include gel electrophoresis (e.g., SDS-PAGE) and liquid chromatography (e.g., high-performance liquid chromatography, HPLC). These techniques enable the isolation of individual proteins from a mixture of protein samples.

 

3. Protein Cleavage

Following separation, proteins are cleaved into smaller peptides, a process achieved through chemical or enzymatic methods. Chemical cleavage methods include the use of cyanogen bromide, while enzymatic cleavage typically employs proteases such as trypsin, chymotrypsin, and elastase.

 

4. Peptide Separation

The peptides resulting from protein cleavage need to be separated for identification. Peptide separation methods primarily involve reverse-phase high-performance liquid chromatography (RP-HPLC) and capillary electrophoresis (CE). These methods facilitate the isolation of individual peptides from a peptide mixture.

 

5. Peptide Identification

Separated peptides are identified using mass spectrometry techniques (e.g., MALDI-TOF MS and ESI-MS). Mass spectrometry accurately determines the molecular weight of peptides and provides their amino acid sequences. By comparing these sequences with known protein sequences in databases, the source proteins can be identified.

 

6. Data Analysis

The final step involves data analysis. Specialized software and databases integrate peptide information obtained from mass spectrometry to reconstruct the complete protein sequence. Bioinformatics methods are essential in this step to ensure the sequencing results' accuracy and reliability.

 

MtoZ Biolabs provides integrate protein sequencing service.