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

Abstract

AbstractWe 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, sensitive and high-throughput N-terminomic profiling without sacrificing the high 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 present a simple and comprehensive N-and C-terminomics platform employing 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 coverages.