Overview of Protein Sequencing: Advantages and Limitations
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Including high-resolution mass spectrometry platforms (e.g., Orbitrap Exploris, Fusion Lumos) for precise qualitative and quantitative analyses of complex samples
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Automated pre-processing platforms to enhance sample consistency and reproducibility
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Dedicated enrichment modules for targeted detection of PTMs (such as phosphorylation, acetylation, etc.)
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Comprehensive data analysis strategies, supporting both database-dependent identification and de novo sequencing
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Integration with transcriptomics, metabolomics, and chemoproteomics to establish a comprehensive functional omics framework
Proteins act as the primary executors of biological functions, with their structure and functionality intricately dependent on the precise arrangement of amino acid sequences. Protein sequencing technology serves as a crucial bridge linking genomics and phenotypic research, advancing our comprehension of biological systems. Utilizing high-resolution mass spectrometry, protein sequencing enables comprehensive qualitative and quantitative characterization of proteins by analyzing peptide fragmentation spectra, while simultaneously identifying post-translational modifications (PTMs). Nevertheless, this technology faces several challenges, including complex sample preparation, intricate data interpretation, and sensitivity issues.
Technical Routes and Processes of Protein Sequencing
1. Core Steps
(1) Extraction and Quantification of Proteins: Total proteins are extracted from cells, tissues, or biological fluids.
(2) Reduction, Alkylation, and Proteolytic Digestion: Proteins are treated with reagents such as DTT and IAA, followed by enzymatic digestion using trypsin or similar enzymes.
(3) Liquid Chromatography Separation (LC): Peptide mixtures are subjected to gradient separation, thereby reducing the complexity of mass spectrometric analysis.
(4) Mass Spectrometry Detection (LC-MS/MS): Precursor ions are selected via MS1, and fragment ion spectra are obtained through MS2 for sequence reconstruction and identification of modifications.
(5) Bioinformatics Analysis and Database Search: Software platforms such as MaxQuant and PEAKS are utilized for peptide identification and protein inference.
2. Sequencing Strategies
(1) Label-Free: No exogenous labels are introduced, suitable for high-throughput screening.
(2) TMT/iTRAQ: Employ isobaric tags for multiplexed sample labeling, facilitating quantitative comparisons under multiple experimental conditions.
(3) SILAC: Incorporate isotopic labeling during cell culture, achieving high labeling efficiency and consistent endogenous expression profiles.
Core Advantages of Protein Sequencing
1. Integrated Multi-Dimensional Information
Protein sequencing not only identifies the types of proteins present but also concurrently analyzes changes in protein abundance, post-translational modifications (e.g., phosphorylation, acetylation), as well as mutations and alternative splicing isoforms.
2. High Throughput and Comprehensive Analysis
Thousands of proteins can be identified in a single experimental run, providing extensive data support for disease research, drug mechanism studies, and biomarker discovery.
3. Independence from Nucleic Acid Information
Protein sequencing can be applied in organisms lacking reference genomes or exhibiting high genetic variability, making it especially valuable in applications such as antibody development and microbiome research.
Limitations and Challenges of Protein Sequencing
1. Insufficient Sequence Coverage
Macromolecules or hydrophobic proteins often result in reduced coverage due to low enzymatic digestion efficiency and the inherent difficulty of peptide fragment analysis, ultimately compromising the overall identification quality.
2. Difficulty in Identifying Modification Sites
Certain post-translational modifications (PTMs), such as glycosylation and ubiquitination, are challenging to pinpoint accurately because of minor mass differences and complex fragmentation patterns, leading to potential localization errors or missed identifications.
3. Complex Data Processing
The vast number of fragment spectra generated by high-throughput mass spectrometry imposes stringent demands on the accuracy of algorithms, the robustness of bioinformatics pipelines, and the precision of manual curation.
4. High Dependence on Sample Quality
Protein extraction efficiency, concentration stability, and enzymatic digestion completeness all significantly influence sequencing performance, with these factors becoming particularly critical in clinical and tissue-derived samples.
Highlights of MtoZ Biolabs Protein Sequencing Service
MtoZ Biolabs integrates mass spectrometry platforms, bioinformatics, and multi-omics capabilities to deliver high-quality, high-throughput protein sequencing solutions:
Protein sequencing technology, with its advanced capabilities for functional information analysis, offers novel avenues for modern biomedicine and drug discovery. However, to fully realize its potential, it is essential to maintain a rigorous understanding of technical nuances, sample conditions, and data interpretation. MtoZ Biolabs is committed to providing comprehensive and professional protein sequencing solutions to research and industry clients, enabling the acquisition of precise and translatable protein insights from complex biological systems. Contact MtoZ Biolabs to discuss your customized sequencing strategies and service plans.
MtoZ Biolabs, an integrated chromatography and mass spectrometry (MS) services provider.
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