Isolation of acetylated and unmodified protein N-terminal peptides by strong cation exchange chromatographic separation of TrypN-digested peptides [Technological Innovation and Resources]

alwin shared this article with you from Inoreader Isolation of acetylated and unmodified protein N-terminal peptides by strong cation exchange chromatographic separation of TrypN-digested peptides [Technological Innovation and Resources] Via MCP Papers in Press 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 pr oteins 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. View on the web Inoreader. Take back control of your news feed. Follow us on Twitter and Facebook.