Can Mass Spectrometry be Used for Protein Sequencing?
Protein sequencing directly reveals the amino acid sequence of proteins, which is essential for understanding their structure and function. With the amino acid sequence determined, the three-dimensional structure of the protein can be predicted, which facilitates the understanding of its biological roles and interaction mechanisms. This is crucial for studying biological processes and disease mechanisms. Current protein sequencing technologies include de novo mass spectrometry sequencing, Edman degradation, and nanopore sequencing. Among these, mass spectrometry-based de novo protein sequencing is the most widely used. This technique does not rely on pre-existing sequence databases, enabling the direct identification of new proteins. De novo sequencing is particularly valuable for discovering novel proteins, such as antibodies, which are critical for immune function and are important tools in medical diagnostics and therapy.
Analysis Workflow
Mass spectrometry-based protein sequencing involves the use of a mass spectrometer to measure the mass of peptide fragments, which correspond to their amino acid sequences, through secondary mass spectra. Peptide sequences are derived from the secondary spectra using de novo sequencing algorithms, and after assembling these sequences, the complete protein sequence is obtained. The key steps in protein sequencing by mass spectrometry are as follows:
1. Sample Preparation
2. Protein Digestion
3. Peptide Separation
4. Mass Spectrometry Analysis
5. Data Analysis
Important considerations include: (1) minimizing protein degradation during sample preparation; (2) selecting the appropriate enzymes for digestion; (3) ensuring stable performance of the mass spectrometer to improve data accuracy.
Case Study
Monoclonal Gammopathy of Undetermined Significance (MGUS) is a plasma cell disorder characterized by the presence of a monoclonal antibody (M-protein) in serum. The authors used liquid chromatography-mass spectrometry (LC-MS) to analyze IgG1-type M-protein in human serum. Various proteases were used for digestion, and de novo sequencing was applied to determine the peptide sequences of the antibody. After assembling these sequences, the complete heavy and light chain sequences of the M-protein were identified. Additionally, glycosylation levels in the antibody's CDR regions were characterized.
Mass spectrometry-based protein sequencing is also instrumental in identifying new drug targets, providing key insights for drug design and screening. By sequencing proteins associated with diseases, scientists can design targeted therapies. Moreover, protein sequencing allows clinicians to assess the disease status of patients more accurately and predict disease progression, which leads to more personalized treatment plans. This not only improves treatment outcomes but also reduces side effects and enhances patients' quality of life.
MtoZ Biolabs has developed software for antibody sequence analysis, integrating mass spectrometry data processing methods, and applying a "wide-in, narrow-out" approach to ensure accurate identification of antibody and protein sequences while preserving all relevant data. The sequencing process uses the highest-resolution and sensitivity mass spectrometers, such as Orbitrap Eclipse and Orbitrap Fusion Lumos, ensuring high-quality data and precise results. The protein/antibody sequencing project typically takes 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." 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." Nature Biotechnology, 2008, 26(12): 1336-1338.
MtoZ Biolabs, an integrated chromatography and mass spectrometry (MS) services provider.
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