Direct Protein Sequencing
Direct protein sequencing is a technique used to determine the primary structure of proteins by analyzing their amino acid sequences. Unlike genome- or transcriptome-based approaches, it directly characterizes protein sequences without relying on genetic information. Traditional protein sequencing methods have primarily relied on DNA or mRNA sequencing to infer protein sequences. However, due to post-transcriptional modifications, alternative splicing, and non-canonical translation, genomic data do not always accurately represent native protein sequences. Direct protein sequencing overcomes this limitation by directly determining protein structures, thereby providing more precise sequence information.
This method is indispensable in proteomics research and is widely applied in novel protein identification, post-translational modification (PTM) analysis, antibody sequencing, and protein mutation studies. In biomedical research, direct protein sequencing plays a critical role in discovering novel biomarkers and advancing precision medicine. For instance, in cancer research, tumor-specific protein variants may serve as potential diagnostic or therapeutic targets. Direct protein sequencing allows precise identification of these variants, providing essential data for personalized medicine. In antibody drug development, this technique is fundamental for determining heavy and light chain sequences. Since the variable region of an antibody dictates its specificity and affinity, accurate sequence determination is crucial for optimizing biologics. Direct protein sequencing facilitates the characterization of amino acid variations, PTMs, and glycosylation sites in antibodies, thereby supporting quality control in biopharmaceutical development.
The core technologies in direct protein sequencing include Edman degradation and mass spectrometry (MS)-based sequencing. Edman degradation is a classical chemical method that sequentially removes amino acids from the N-terminus of a peptide for identification, making it particularly suitable for sequencing short peptides and purified proteins. However, its time-consuming nature and limited capability for long peptides have restricted its application in high-throughput proteomics. In contrast, mass spectrometry-based sequencing has emerged as the predominant approach in modern direct protein sequencing. By analyzing the mass-to-charge ratio (m/z) of fragmented peptides, this technique reconstructs amino acid sequences without relying on genomic information.
Mass spectrometry-based direct protein sequencing typically employs tandem mass spectrometry (MS/MS), with common approaches including proteolytic digestion combined with mass spectrometry (e.g., Lys-C and trypsin digestion) and de novo sequencing. De novo sequencing, which derives protein sequences directly from mass spectrometry data without a reference database, is particularly valuable for identifying novel proteins, sequencing antibody variable regions, detecting protein mutations, and characterizing PTMs. Additionally, the integration of high-resolution mass spectrometry (e.g., Orbitrap, TOF-MS) with advanced fragmentation techniques (e.g., HCD, ETD, EAD) has significantly improved the accuracy and coverage of direct protein sequencing, enabling the analysis of complex protein samples.
MtoZ Biolabs offers high-precision protein sequencing services tailored for applications such as antibody drug development, protein mutation studies, and novel protein identification. By leveraging advanced mass spectrometry techniques, our expert team ensures precise and reliable sequence determination, supporting high-quality research and biopharmaceutical development.
MtoZ Biolabs, an integrated chromatography and mass spectrometry (MS) services provider.
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