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

Paul J Boersema1, Nadia Taouatas, A F Maarten Altelaar

  • 1Biomolecular Mass Spectrometry and Proteomics Group, Utrecht Institute for Pharmaceutical Sciences and Bijvoet Center for Biomolecular Research, Utrecht University, The Netherlands.

Molecular & Cellular Proteomics : MCP
|December 2, 2008
PubMed

Insights

Lys-N enzyme combined with strong cation exchange enrichment and MALDI-MS/MS analysis offers a new proteomics strategy. This method enables straightforward de novo sequencing and protein identification, proving valuable for research.

Area of Science:

  • Proteomics
  • Mass Spectrometry
  • Enzymology

Background:

  • Lys-N (Lysine-specific endopeptidase N) is a metalloendopeptidase.
  • Matrix-assisted laser desorption/ionization tandem mass spectrometry (MALDI-MS/MS) is a key proteomics technique.
  • De novo sequencing aims to determine protein sequences directly from mass spectrometry data.

Purpose of the Study:

  • To evaluate the potential of Lys-N for MALDI-MS/MS proteomics.
  • To assess Lys-N's utility in de novo sequencing applications.
  • To establish a novel proteomics workflow combining Lys-N, enrichment, and MALDI-MS/MS.

Main Methods:

  • Digestion of HEK293 cellular lysate and ostrich muscle tissue lysate with Lys-N and Lys-C.
  • Peptide enrichment using strong cation exchange chromatography.
  • MALDI-MS/MS analysis of enriched peptides.
  • In-gel digestion of a 42 kDa protein band from ostrich muscle.

Main Results:

  • Lys-N digestion yielded peptides with clear b-ion sequence ladders in CID spectra.
  • De novo sequencing successfully identified creatine kinase and actin from ostrich muscle.
  • The combined method proved effective for protein identification.

Conclusions:

  • Lys-N is a valuable enzyme for MALDI-MS/MS proteomics.
  • The integration of Lys-N, strong cation exchange, and MALDI-MS/MS provides an effective strategy for de novo sequencing.
  • This approach offers a promising alternative for proteomics research.

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