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Mol Cell Proteomics. 2009 Apr;8(4):650-60. doi: 10.1074/mcp.M800249-MCP200. Epub 2008 Nov 29.

Straightforward and de novo peptide sequencing by MALDI-MS/MS using a Lys-N metalloendopeptidase.

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Biomolecular Mass Spectrometry and Proteomics Group, Utrecht Institute for Pharmaceutical Sciences and Bijvoet Center for Biomolecular Research, Utrecht University, The Netherlands.


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.

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