iNrich, a rapid and robust method to enrich N-terminal proteome in a highly multiplexed platform.

Terminal proteomics is limited in that it is optimized for large-scale analysis via multistep processes involving liquid chromatography. Here, we present an integrated N-terminal peptide enrichment method (iNrich) that can handle as little as 25 microg of cell lysate via a single-stage encapsulated solid-phase extraction column. iNrich enables simple, rapid and reproducible sample processing, treatment of a wide range of protein amount (25 microg ~ 1 mg), multiplexed parallel sample preparation, and in-stage sample prefractionation using a mixed anion exchange filter. We identified ~5,000 N-terminal peptides (Nt-peptides) from only 100 microg human cell lysate including Nt-formyl peptides. Multiplexed sample preparation facilitated quantitative and robust enrichment of N-terminome with dozens of samples simultaneously. We further developed the method to incorporate isobaric tags such as tandem mass tag (TMT) and used it to discover novel peptides during ER stress analysis. The iNrich facilitated high-throughput N-terminomics and degradomics at low cost using commercially available reagents and apparatus, without requiring arduous procedures.