Isolation of acetylated and unmodified protein N-terminal peptides by strong cation exchange chromatographic separation of TrypN-digested peptides.

Isolation of acetylated and unmodified protein N-terminal peptides by strong cation exchange chromatographic separation of TrypN-digested peptides. Mol Cell Proteomics. 2020 Nov 02;: Authors: Chang CH, Chang HY, Rappsilber J, Ishihama Y Abstract 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, 1,550 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. Since 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. PMID: 33139343 [PubMed - as supplied by publisher]
Source: Molecular and Cellular Proteomics : MCP - Category: Molecular Biology Authors: Tags: Mol Cell Proteomics Source Type: research