Straightforward and de novo peptide sequencing by MALDI-MS/MS using a Lys-N metalloendopeptidase.
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.
Boersema
Paul J
PJ
Biomolecular Mass Spectrometry and Proteomics Group, Utrecht Institute for Pharmaceutical Sciences and Bijvoet Center for Biomolecular Research, Utrecht University, The Netherlands.
Taouatas
Nadia
N
Altelaar
A F Maarten
AF
Gouw
Joost W
JW
Ross
Philip L
PL
Pappin
Darryl J
DJ
Heck
Albert J R
AJ
Mohammed
Shabaz
S
D000199:Actins / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002645:Chickens; D003402:Creatine Kinase / Q000737:chemistry; D006801:Humans; D008239:Lysine / Q000378:metabolism*; D008666:Metalloendopeptidases / Q000378:metabolism*; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry*; D011392:Proline / Q000378:metabolism; D020539:Sequence Analysis, Protein / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D020310:Struthioniformes; D053719:Tandem Mass Spectrometry
D000199:Actins; D010455:Peptides; D011392:Proline; D003402:Creatine Kinase; D008666:Metalloendopeptidases; D008239:Lysine