Boersema, P. J., Taouatas, N., Altelaar, A. F. M., Gouw, J. W., Ross, P. L., Pappin, D. J., Heck, A. J. R., Mohammed, S. (April 2009) Straightforward and de Novo Peptide Sequencing by MALDI-MS/MS Using a Lys-N Metalloendopeptidase. Molecular & Cellular Proteomics, 8 (4). pp. 650-660. ISSN 1535-9476
Abstract
In this work, we explore the potential of the metalloendopeptidase Lys-N for MALDI-MS/MS proteomics applications. Initially we digested a HEK293 cellular lysate with Lys-N and, for comparison, in parallel with the protease Lys-C. The resulting peptides were separated by strong cation exchange to enrich and isolate peptides containing a single N-terminal lysine. MALDI-MS/MS analysis of these peptides yielded CID spectra with clear and often complete sequence ladders of b-ions. To test the applicability for de novo sequencing we next separated an ostrich muscle tissue protein lysate by one-dimensional SDS-PAGE. A protein band at 42 kDa was in-gel digested with Lys-N. Relatively straightforward sequencing resulted in the de novo identification of the two ostrich proteins creatine kinase and actin. We therefore conclude that this method that combines Lys-N, strong cation exchange enrichment, and MALDI-MS/MS analysis provides a valuable alternative proteomics strategy. Molecular & Cellular Proteomics 8:650-660, 2009.
| Item Type: | Paper |
|---|---|
| Uncontrolled Keywords: | POST-SOURCE DECAY TIME-OF-FLIGHT TANDEM MASS-SPECTROMETRY PROTONATED PEPTIDES PROTEIN IDENTIFICATION COMPARATIVE PROTEOMICS CHARGE DERIVATIZATION TRYPTIC PEPTIDES FRAGMENTATION DISSOCIATION |
| Subjects: | bioinformatics > genomics and proteomics Investigative techniques and equipment bioinformatics > genomics and proteomics > genetics & nucleic acid processing > protein structure, function, modification Investigative techniques and equipment > spectroscopy > mass spectrometry bioinformatics > genomics and proteomics > genetics & nucleic acid processing > protein structure, function, modification > protein characterization Investigative techniques and equipment > spectroscopy |
| CSHL Authors: | |
| Communities: | CSHL labs > Pappin lab |
| Depositing User: | Matt Covey |
| Date: | April 2009 |
| Date Deposited: | 19 Feb 2013 19:18 |
| Last Modified: | 19 Feb 2013 19:18 |
| PMCID: | PMC2667348 |
| Related URLs: | |
| URI: | https://repository.cshl.edu/id/eprint/27462 |
Actions (login required)
 |
Administrator's edit/view item |