Methods and Applications for Protein Sequence Determination

Protein sequence determination is the process of determining the exact order of amino acids in proteins. It enables the analysis of amino acid sequences, confirmation of protein structure and function, identification of novel protein biomarkers and drug targets, construction of phylogenetic trees to understand evolutionary relationships, and identification of orthologous and paralogous proteins.

 

Protein sequencing can be divided into three main methods: studying the N-terminus, exploring the C-terminus (methods are limited, often involving carboxypeptidase), and breaking down peptides into peptides. Classical protein sequencing methods such as Edman degradation can systematically remove the amino acid residues at the peptide amino terminus, thus maintaining the integrity of the protein. In contrast, next-generation protein sequencing (NGPS) uses advanced technology to comprehensively identify all types of proteins in biological organisms, providing unprecedented precision and potential applications for disease research and immunotherapy.

 

Edman Degradation

This is the most traditional method, which can sequentially and one by one determine the N-terminal amino acid sequence of proteins. In each round, the N-terminal amino acid of the protein is selectively marked, then cut off and identified. This process can be repeated many times to gradually determine the amino acid sequence. The disadvantage is that it can usually only determine the first 20-30 amino acid residues of the protein and requires a large amount of pure protein.

 

1. Applications

(1) N-Terminal Sequence Analysis

(2) Sequence Analysis of Small to Medium-Sized Peptides (Usually 5 to 50 Amino Acids Long)

 

Mass Spectrometry

In recent years, mass spectrometry has become the preferred method for protein sequencing, which involves ionization, charged particle acceleration into the mass analyzer, and detection based on deflection proportional to the mass-to-charge ratio (m/z). For large proteins, sample introduction methods include diffusion, liquid injection, or laser desorption. LCMS, which combines HPLC with mass spectrometry, is used for the analysis of complex mixtures.

 

LC-MS is currently a commonly used technique, which uses electrospray ionization (ESI) to achieve a reliable interface. It is suitable for various biomolecules, tandem MS and stable isotopes can improve sensitivity and accuracy. Although method optimization is required to reduce ion suppression, rapid scanning can achieve multiple analyses, measuring multiple compounds in a single run. As instruments become cheaper, LC-MS is becoming increasingly important in clinical biochemistry, competing with traditional methods such as liquid chromatography and immunoassays.

 

Quadrupole mass analyzers are typically used for tandem mass spectrometry (MS/MS), selecting ions based on different m/z ratios, and using collision-induced dissociation (CID) to fragment selected ions in the ion trap, thus allowing for the determination of peptide/protein sequences. This technique usually involves two mass analyzers separated by a collision cell, but a quadrupole ion trap can also be used to complete it in a single mass analyzer.

 

1. Applications

(1) Terminal (N or C Terminal) Sequencing, Full Sequence Determination, De Novo Sequencing and Mutation Analysis of Proteins

 

(2) Identification of Unknown Proteins

The peptide mass and sequence information obtained by mass spectrometric analysis are matched with the protein database to identify the proteins in the sample.

 

(3) Identification of Proteins in Complex Samples

Identifying hundreds to thousands of proteins from fluid, cell, or tissue samples.

 

Next-Generation Sequencing Technology

Next-Generation Protein Sequencing (NGPS) has completely changed the comprehensive identification of all types of proteins in a single biological organism. It represents a technical initiative aimed at accurately extracting proteome data. NGPS can identify low-abundance proteins, including those that undergo post-translational modifications. NGPS is expected to completely change disease research, immunotherapy, and vaccine development by precisely identifying protein sequences and enabling antibody variability studies.

 

1. Applications

(1) High-Throughput Sequencing

NGPS can achieve high-throughput protein sequencing, greatly improving the speed and efficiency of sequencing. This is very useful for handling large-scale sample collections or research projects that require a large amount of protein analysis.

 

(2) Low-Cost Sequencing

The development of NGPS technology can reduce the cost of protein sequencing, enabling more research teams and laboratories to undertake proteomics research, promoting the development of this field.

 

(3) Deep Sequencing

NGPS can achieve deeper protein sequencing, allowing scientists to more comprehensively understand the protein composition and characteristics in the sample. This is very important for studying complex biological systems, disease mechanisms, and drug action mechanisms.

 

(4) Single-Cell Proteomics

The development of NGPS technology has made single-cell proteomics research possible. Scientists can use NGPS to sequence proteins in individual cells, revealing protein expression patterns and functions in different cell states.

 

(5) Dynamic Proteomics

NGPS can achieve dynamic monitoring of the proteome, including post-translational modifications of proteins, changes in protein interaction networks, etc. This helps to understand the regulatory mechanisms of cellular and extracellular environments on protein expression and function.

 

MtoZ Biolabs uses nano LC-MS/MS nanoscale chromatography combined with tandem mass spectrometry and Shimadzu's Edman degradation sequencing system to analyze protein sequences, providing services including analysis of protein amino acid composition, N-terminal sequencing, C-terminal sequencing, full-length sequence analysis, and Edman degradation-based protein N-terminal sequence analysis. For proteins with unknown theoretical sequences, de novo protein sequencing services are provided to analyze protein sequences.