Straightforward and de novo peptide sequencing by MALDI-MS/MS using a Lys-N metalloendopeptidase.
Mol Cell Proteomics
; 8(4): 650-60, 2009 Apr.
Article
en En
| MEDLINE
| ID: mdl-19043101
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.
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Péptidos
/
Metaloendopeptidasas
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Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción
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Análisis de Secuencia de Proteína
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Lisina
Límite:
Animals
/
Humans
Idioma:
En
Revista:
Mol Cell Proteomics
Asunto de la revista:
BIOLOGIA MOLECULAR
/
BIOQUIMICA
Año:
2009
Tipo del documento:
Article
País de afiliación:
Países Bajos
Pais de publicación:
Estados Unidos