Straightforward and de Novo Peptide Sequencing by MALDI-MS/MS Using a Lys-N Metalloendopeptidase

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

URL: http://www.ncbi.nlm.nih.gov/pubmed/19043101
DOI: 10.1074/mcp.M800249-MCP200

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: http://repository.cshl.edu/id/eprint/27462

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