Isolation of Acetylated and Unmodified Protein N-Terminal Peptides by Strong Cation Exchange Chromatographic Separation of TrypN-Digested Peptides.
Mol Cell Proteomics
; 20: 100003, 2021.
Article
em En
| MEDLINE
| ID: mdl-33517145
We developed a simple and rapid method to enrich protein N-terminal peptides, in which the protease TrypN is first employed to generate protein N-terminal peptides without Lys or Arg and internal peptides with two positive charges at their N termini, and then, the N-terminal peptides with or without N-acetylation are separated from the internal peptides by strong cation exchange chromatography according to a retention model based on the charge/orientation of peptides. This approach was applied to 20 µg of human HEK293T cell lysate proteins to profile the N-terminal proteome. On average, 1550 acetylated and 200 unmodified protein N-terminal peptides were successfully identified in a single LC/MS/MS run with less than 3% contamination with internal peptides, even when we accepted only canonical protein N termini registered in the Swiss-Prot database. Because this method involves only two steps, protein digestion and chromatographic separation, without the need for tedious chemical reactions, it should be useful for comprehensive profiling of protein N termini, including proteoforms with neo-N termini.
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Base de dados:
MEDLINE
Assunto principal:
Peptídeos
/
Tripsina
Limite:
Humans
Idioma:
En
Ano de publicação:
2021
Tipo de documento:
Article