One-step N-Terminomics Based on Isolation of Protein N-Terminal Peptides From LysargiNase Digests by Tip-Based Strong Cation Exchange Chromatography.

Kazuya Morikawa; Hiroshi Nishida; Koshi Imami; Yasushi Ishihama

doi:10.1016/j.mcpro.2024.100820

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One-step N-Terminomics Based on Isolation of Protein N-Terminal Peptides From LysargiNase Digests by Tip-Based Strong Cation Exchange Chromatography.

Kazuya Morikawa·Hiroshi Nishida·Koshi Imami·Yasushi Ishihama

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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, sensitive, reproducible, 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.

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Humans Chromatography, Ion Exchange HEK293 Cells Proteomics Peptides Proteome LysargiNase StageTip protein terminal peptides strong cation exchange chromatography Journal Article Research Support, Non-U.S. Gov't

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Kazuya Morikawa, Hiroshi Nishida, Koshi Imami, Yasushi Ishihama. 2024-07-26. One-step N-Terminomics Based on Isolation of Protein N-Terminal Peptides From LysargiNase Digests by Tip-Based Strong Cation Exchange Chromatography.. https://doi.org/10.1016/j.mcpro.2024.100820

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