Straightforward and de novo peptide sequencing by MALDI-MS/MS using a Lys-N metalloendopeptidase
| Autor: | Albert J. R. Heck, A. F. Maarten Altelaar, Paul J. Boersema, Joost W. Gouw, Darryl J. Pappin, Philip L. Ross, Nadia Taouatas, Shabaz Mohammed |
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| Jazyk: | angličtina |
| Rok vydání: | 2016 |
| Předmět: |
Proline
Sequence analysis Molecular Sequence Data Biology Tandem mass spectrometry Proteomics Biochemistry Analytical Chemistry Cell Line Peptide mass fingerprinting Sequence Analysis Protein Tandem Mass Spectrometry Animals Humans Amino Acid Sequence Molecular Biology Peptide sequence Creatine Kinase Struthioniformes Research Lysine Metalloendopeptidases De novo peptide sequencing Lys-N Molecular biology Actins Spectrometry Mass Matrix-Assisted Laser Desorption-Ionization Metalloendopeptidase Peptides Chickens |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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