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bioRxiv · 10.1101/2020.06.15.153684

Isolation of protein N-terminal peptides by charge-mediated position- elective enrichment using strong cation exchange chromatography

Chang, C.-H.·Chang, H.-Y.·Rappsilber, J.·ishihama, Y.
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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 are separated from the internal peptides by means of CHArge-Mediated Position-selective (CHAMP) enrichment using strong cation exchange (SCX) chromatography. This CHAMP-SCX approach was applied to 20 g 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.

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Chang, C.-H., Chang, H.-Y., Rappsilber, J., ishihama, Y.. 2020-06-16. Isolation of protein N-terminal peptides by charge-mediated position- elective enrichment using strong cation exchange chromatography. https://doi.org/10.1101/2020.06.15.153684

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