Principle and Steps of Protein Sequencing
-
Broad sample compatibility: Supporting sample types including cells, tissues, serum, and exosomes
-
High-sensitivity instrumentation: Orbitrap Exploris, Fusion Lumos, timsTOF, among others
-
Specialized sample preparation: Automated systems for enzymatic digestion, purification, and enrichment
-
Advanced data analysis: Quantitative profiling, pathway enrichment analysis, and protein-protein interaction networks
-
Multi-omics integration: Compatible with transcriptomic, metabolomic, and chemical proteomic analyses
Proteins play direct and essential roles in biological processes, and their types, abundances, and post-translational modification states directly determine cellular function. In contrast to DNA sequencing, which focuses on the “potential coding information,” protein sequencing aims to uncover the “actual expression and functional states” of proteins. By precisely identifying protein sequences and their modification patterns, researchers can gain comprehensive insights into cellular behavior, disease mechanisms, and drug responses. Among available techniques, mass spectrometry (MS) serves as the cornerstone of protein sequencing, with liquid chromatography-tandem mass spectrometry (LC-MS/MS) emerging as the predominant platform in current proteomic research.
Basic Principle of Protein Sequencing
The fundamental objective of protein sequencing is to deduce the amino acid sequence of peptide fragments by measuring their mass and fragmentation patterns using mass spectrometry. The sequencing process typically involves the following key steps:
1. Enzymatic Digestion to Generate Peptides
Target proteins are enzymatically cleaved into stable and analyzable peptide fragments using proteases such as trypsin.
2. Chromatographic Separation
The resulting complex peptide mixture is separated using high-performance liquid chromatography (LC) to enhance analytical resolution and detection sensitivity.
3. Mass Spectrometry Analysis
Peptides are introduced into the mass spectrometer, where their mass-to-charge ratios are first measured in the MS1 scan. Selected peptides are then subjected to tandem MS (MS2) for fragmentation analysis, enabling the characterization of their amino acid sequences.
4. Database Matching
The acquired fragmentation spectra are compared against protein databases to identify the peptide sequences and their corresponding proteins.
By statistically analyzing the frequency, intensity, and post-translational modifications of peptides across the entire sample, researchers can perform both qualitative identification and quantitative assessment of proteins.
Main Steps of Protein Sequencing
Step 1: Protein Extraction and Quantification
Total proteins are extracted from cells, tissues, or body fluids. The concentration is determined using standard assays such as the BCA or Bradford method, providing a consistent baseline for downstream processing.
Step 2: Protein Reduction, Alkylation, and Enzymatic Digestion
Disulfide bonds are reduced using dithiothreitol (DTT), and free sulfhydryl groups are alkylated with iodoacetamide (IAA), facilitating the unfolding of protein tertiary structure. Subsequently, the proteins are digested with trypsin or other specific proteases to generate peptides suitable for mass spectrometry analysis.
Step 3: Polypeptide Purification and Fractionation
To reduce sample complexity and enhance both mass spectrometry performance and data coverage, solid-phase extraction (e.g., C18 columns) or high-pH reverse-phase fractionation can be employed.
Step 4: LC-MS/MS Detection
Following chromatographic separation, polypeptides are introduced into the mass spectrometer for MS1 and MS2 acquisition, generating a comprehensive mass spectrometry dataset.
Step 5: Data Analysis and Protein Identification
Software platforms such as MaxQuant and Proteome Discoverer are utilized for database searching, peptide identification and protein quantification, differential expression analysis, and functional annotation including GO and KEGG pathways.
Selection of Protein Sequencing Strategies: Emphasizing Both Quantification and Post-Translational Modifications
Depending on the specific research objectives, various protein sequencing strategies can be selected:
1. Label-Free
Does not require chemical labeling; procedurally straightforward, suitable for large-cohort studies or early-stage screening.
2. TMT/iTRAQ
Isobaric labeling methods suitable for quantitative comparisons across multiple experimental groups, with high analytical sensitivity.
3. SILAC
Stable isotope labeling by amino acids in cell culture, suitable for endogenous labeling in cellular systems.
4. PTM Analysis
Specialized in the detection and quantification of post-translational modifications such as phosphorylation and acetylation.
MtoZ Biolabs offers customized proteomics strategies tailored to specific project requirements, ensuring an optimal balance between proteome coverage, quantitative accuracy, and biological depth.
Advantages of MtoZ Biolabs in Protein Sequencing Services
As a leading proteomics service provider, MtoZ Biolabs has established a comprehensive technical workflow and state-of-the-art mass spectrometry platforms for protein sequencing:
As a core approach to elucidating functional protein states, protein sequencing is transitioning from basic research into applications such as disease diagnostics, target discovery, and drug mechanism studies. Leveraging cutting-edge mass spectrometry technologies and customizable workflows, MtoZ Biolabs provides researchers with high-quality, high-throughput, and interpretable proteomics data. Researchers are encouraged to consult MtoZ Biolabs for tailored protein sequencing solutions to advance their scientific investigations.
MtoZ Biolabs, an integrated chromatography and mass spectrometry (MS) services provider.
Related Services
How to order?
