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

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, 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. ### Competing Interest Statement The authors have declared no competing interest. * SCX : strong cation exchange CHAMP-N : chromatographic amplification of protein N-terminal peptides ACN : acetonitrile COFRADIC : combined fractional diagonal chromatography TAILS : terminal amine isotopic labeling of substrates CHAMP-C : chromatographic amplification of protein C-terminal peptides PTS : phase-transfer surfactants SDC : sodium deoxycholate SLS : sodium N -lauroylsarcosinate Tris−HCl : tris(hydroxymethyl)aminomethane hydrochloride TFA : trifluoroacetic acid AGC : automatic gain control CV : compensation voltage FA : formic acid iMet : initiator methionine CHAMP-NC : chromatographic amplification of protein N- and C-terminal peptides MOLEX : metal oxide-based ligand-exchange Nt-peptide : N-terminal peptide.