One-step N-terminomics based on isolation of protein N-terminal peptides from LysargiNase digests by tip-based strong cation exchange chromatography

Abstract

We have developed a one-step isolation method for protein N-terminal peptides from LysargiNase digests by pipette tip-based strong cation exchange (SCX) chromatography. This CHAMP-N (CHromatographic AMplification of Protein N-terminal peptides) method using disposable and parallel-processable SCX tips instead of conventional HPLC SCX columns facilitates simple, sensitivie and high-throughput N-terminomic profiling without sacrificing identification numbers and selectivity achieved by the HPLC-based method. By applying the CHAMP-N method to HEK293T cells, we identified novel cleavage sites for signal and transit peptides, and non-canonical translation initiation sites. Finally, for proteome-wide terminomics, we have established a simple and comprehensive N- and C-terminomics platform based on three different tip-based approaches including CHAMP-N, in which protease digestion and one-step isolation by tip LC are commonly used to achieve complementary terminome coverage.In BriefLarge-scale isolation of protein N-terminal peptides from LysargiNase digests was achieved by one-step strong cation exchange chromatography with disposable and parallel-processable pipette-tip columns. Novel cleavage sites for signal or transit peptides and non-canonical translation initiation sites were identified by this tip-based method. Together with two other methods similarly consisting of protease digestion and one-step tip LC, we have established a simple and comprehensive N- and C-terminomics platform based on CHAMP (chromatographic amplification of protein terminal peptides) approaches.HighlightsOne-step isolation of protein N-terminal peptides from LysargiNase digests by tip-based strong cation exchange chromatography.Highly efficient isolation of protein N-terminal peptides by tip LC comparable to HPLC.Identifying novel post-translational cleavage sites and non-canonical translation initiation sites.Complementary terminome coverage by the three CHAMP methods consisting of protease digestion and one-step tip LC.