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

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


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.

MeSH terms

  • Actins / chemistry
  • Amino Acid Sequence
  • Animals
  • Cell Line
  • Chickens
  • Creatine Kinase / chemistry
  • Humans
  • Lysine / metabolism*
  • Metalloendopeptidases / metabolism*
  • Molecular Sequence Data
  • Peptides / chemistry*
  • Proline / metabolism
  • Sequence Analysis, Protein / methods*
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization*
  • Struthioniformes
  • Tandem Mass Spectrometry


  • Actins
  • Peptides
  • Proline
  • Creatine Kinase
  • Metalloendopeptidases
  • Lysine