Principle of Protein Full-Length Sequencing

Proteins are fundamental molecules in biological processes, with their structures and functions directly impacting the physiology of organisms. Determining the full-length sequence of proteins is crucial for understanding their functional mechanisms, designing drugs, and studying diseases. Protein full-length sequencing involves accurately determining the amino acid sequence of a protein from the N-terminus to the C-terminus through various technical approaches.

 

Basic Principles of Protein Full-Length Sequencing

Protein full-length sequencing primarily relies on mass spectrometry (MS) and protein chemical degradation techniques. The combination of these technologies overcomes the limitations of individual methods and provides precise protein sequence information.

 

1. Mass Spectrometry

Mass spectrometry is a core tool in protein full-length sequencing. It measures the mass-to-charge ratio (m/z) of ionized proteins or peptides, thereby inferring their molecular weight and structure. Major MS methods include Electrospray Ionization (ESI) and Matrix-Assisted Laser Desorption/Ionization (MALDI), each with distinct advantages.

 

(1) ESI-MS

Ideal for analyzing large proteins and complex mixtures. The sample solution is sprayed to form charged droplets, and as the solvent evaporates, charged protein ions are produced.

 

(2) MALDI-MS

Suitable for analyzing smaller proteins and peptides. The sample, mixed with a matrix, is ionized under laser irradiation to form charged ions.

 

2. Protein Chemical Degradation Techniques

Edman degradation and enzymatic digestion are key methods for protein chemical degradation. These methods progressively cut protein molecules into smaller peptides for subsequent MS analysis.

 

(1) Edman Degradation

Sequentially removes and identifies each amino acid starting from the protein's N-terminus. Although it provides high-precision sequence information, its efficiency is lower for long-chain proteins.

 

(2) Enzymatic Digestion

Uses specific enzymes (e.g., trypsin) to cleave proteins into peptides. The peptides are then separated and identified using Liquid Chromatography (LC) coupled with Mass Spectrometry (LC-MS).

 

3. Data Analysis and Integration

The vast amount of data generated from MS and chemical degradation techniques require analysis and integration using bioinformatics tools. Common software includes Mascot, Sequest, and MaxQuant, which can identify peptide sequences and assemble them into complete protein sequences.

 

MtoZ Biolabs provides integrate protein full-length sequencing service.