What is Protein Sequencing?
Protein sequencing provides direct insights into the amino acid sequences of proteins, a fundamental step for comprehending protein structure and function. Following sequencing, it becomes possible to predict a protein's three-dimensional structure, facilitating the analysis of its biological functions and interaction mechanisms. Such insights are critical for advancing our understanding of life processes and elucidating the origins and progression of diseases. Current protein sequencing methods primarily include mass spectrometry-based de novo sequencing, Edman degradation, and nanopore sequencing, with mass spectrometry-based de novo sequencing being the most commonly employed technique due to its reliability and applicability. De novo protein sequencing, a novel approach that determines amino acid sequences without relying on pre-existing sequence or database information, is especially valuable for identifying new proteins. Antibodies, as essential molecules within the immune system, serve as vital tools in medical diagnosis and therapy; notably, de novo protein sequencing has significant applications within antibody research and development.
Analysis Workflow
Mass spectrometry-based protein sequencing leverages a mass spectrometer to acquire mass-to-charge (m/z) information from peptide fragments, generating tandem mass spectra (MS/MS) that contain the amino acid sequence data of peptides. Using de novo sequencing algorithms, these peptide sequences are directly interpreted from MS/MS spectra and subsequently assembled to reconstruct the complete amino acid sequence of the protein. The technical workflow for mass spectrometry-based protein sequencing comprises the following steps: sample preparation, protein digestion, peptide separation, mass spectrometry detection, and data analysis. Key considerations for mass spectrometry-based protein sequencing include: ① minimizing protein degradation during sample preparation; ② selecting appropriate enzymes for efficient protein digestion; and ③ maintaining stable performance of the mass spectrometer to enhance data accuracy.
Case Study
Monoclonal gammopathy of undetermined significance (MGUS) is a plasma cell disorder marked by the presence of a dominant monoclonal antibody, referred to as M protein, in serum. In this study, the authors identified IgG1-type M protein in human serum using liquid chromatography-tandem mass spectrometry (LC-MS/MS). To enhance sequence coverage, multiple proteases were employed to digest M protein, and de novo sequencing methods were used to analyze the resulting antibody peptides. Through peptide assembly, the full-length sequences of the M protein's heavy and light chains were determined. The authors further characterized glycosylation patterns within the antibody's complementarity-determining regions (CDRs).
Protein sequencing facilitates the identification of novel drug targets, providing essential insights for drug design and screening. With sequencing technology, scientists can pinpoint proteins associated with specific diseases, paving the way for the development of targeted therapies. Moreover, protein sequencing enables clinicians to evaluate disease status and predict disease progression with greater accuracy, which supports the creation of more personalized treatment regimens. Such approaches not only optimize therapeutic outcomes but also minimize adverse effects, ultimately enhancing patients' quality of life.
MtoZ Biolabs has developed advanced antibody sequence analysis software, informed by research published in Nature Biotechnology and the Journal of Proteome Research (JPR) and incorporating domain-specific expertise. This software employs a proprietary algorithm for processing raw mass spectrometry data, adhering to a broad-input, selective-output model to guarantee comprehensive and precise antibody/protein sequence identification. The anticipated timeline for protein or antibody sequencing projects is approximately 3 to 4 weeks.
References
[1] albertj,denboer,arjand,et al.Direct Mass Spectrometry-Based Detection and Antibody Sequencing of Monoclonal Gammopathy of Undetermined Significance from Patient Serum: A Case Study[J].Journal of Proteome Research, 2023, 22(9):3022-3028.
[2] Bandeira N, Pham V, Pevzner P, et al. Automated de novo protein sequencing of monoclonal antibodies [J]. Nature Biotechnology, 2008, 26(12):1336-1338
MtoZ Biolabs, an integrated chromatography and mass spectrometry (MS) services provider.
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