| Status | 已發表Published |
| A New Method for Protein Sequence Analysis and PTM Characterization with 100% Sequence Coverage | |
Ang, L.; Poon, C. W.
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| 2019-06-12 | |
| Source Publication | 6th Macau Symposium on Biomedical Sciences 2019
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| Abstract | In the field of proteomics, protein identification and characterization of post-translational modifications (PTMs) require proteolysis of proteins into peptides for fragmentation by tandem mass spectrometry followed by computational mapping of peptide sequences. Trypsin is the most commonly used protease though it provides limited protein sequence coverage owing to its substrate specificity to lysine and arginine. The limited protein sequence coverage issue can be solved by undertaking an approach involving multiple independent enzymatic digestions with two or more digestive enzymes that cleave at different residues to generate peptides which contains overlapping amino acid sequences across the protein sequence. Unfortunately, such approach is tedious, time consuming, and substantially increases the amount of protein required for analysis of protein sequence and PTMs. In this study, we developed a rapid (≤1 hour) proteolytic method to generate overlapping peptides that enabled 100% protein sequence coverage of 17 proteins, sequence variant identification and comprehensive modification characterization. Up to 99.9% protein sequence coverage was obtained when the method was applied to sequence a 6-protein mixture. Moreover, protein PTMs, such as phosphorylation, glycosylation and protein N-terminal modification, could be characterized at the same time. For E. coli proteome, we obtained 100% (≥90%) protein sequence coverage for 113 (340) proteins and identified all known and 36 novel N-terminal acetylated proteins with confidence. Taken together, the results establish the utility of our new method for various applications in bottom-up proteomics. |
| Keyword | proteomics mass spectrometry post-translational modification protein sequence coverage |
| Language | 英語English |
| The Source to Article | PB_Publication |
| PUB ID | 48698 |
| Document Type | Conference paper |
| Collection | DEPARTMENT OF BIOMEDICAL SCIENCES |
| Corresponding Author | Poon, C. W. |
| Recommended Citation GB/T 7714 | Ang, L.,Poon, C. W.. A New Method for Protein Sequence Analysis and PTM Characterization with 100% Sequence Coverage[C], 2019. |
| APA | Ang, L.., & Poon, C. W. (2019). A New Method for Protein Sequence Analysis and PTM Characterization with 100% Sequence Coverage. 6th Macau Symposium on Biomedical Sciences 2019. |
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