Development of a Rapid and Robust Method to Enrich N-terminal Proteome in a Highly Multiplexed Platform

Abstract

A coding sequence in genome is a blueprint of a protein, however, N-terminus of a protein may be changed during translation, maturation, and even after transferred to designated site. Mature protein’s N-terminus may alter e.g. by post translational modification or by signal peptide removal. Additionally, some proteases could cleave proteins and often C-terminal fragment act as a signal stimulus. To find alterations N-termini of certain biological systems, N-terminal proteomics methods have been developed to analyze the N-terminal peptides qualitatively or quantitatively or both. Most N-terminomic methods include two major steps. Labeling step and depletion step are essential which is to label N-termini of proteins and to deplete non-N-terminal peptides, respectively. N-terminomic methods are limited in that they are optimized for large-scale analysis via multistep processes involving liquid chromatography. Since many steps could lead to loss of analytes, i.e. N-terminal peptides, N-terminomic methods are indeed fit to use large amount of input materials like milligram scale. Putting large masses to process can mitigate the losses during N-terminome enrichment, however, it restricts the subject of study to have plenty extent. Here, I present an integrated N-terminal peptide enrichment method (iNrich) that can handle as little as 25 µg 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 µg ~ 1 mg), multiplexed parallel sample preparation, and in-stage sample prefractionation using a mixed anion exchange filter. I identified ~5,000 N-terminal peptides (Nt-peptides) from only 100 µg human cell lysate including Nt-formyl peptides. To validate the enriched resultant, I acquired N-terminal peptides from known N-terminal acetyltransferase (NAT) deletion mutant in Saccharomyces cerevisiae, as expected, N-terminal peptides enriched by iNrich showed alterations on their N-terminal acetylation in various NAT mutants. In addition, 18-plex multiplexed sample preparation using triplicate enrichment of various cell lines provided the evidence that iNrich could facilitate quantitative and robust enrichment of N-terminome with dozens of samples simultaneously. I further developed the method to incorporate isobaric tags such as tandem mass tag (TMT) and used it to discover novel peptides during ER stress induced systems and to identify differences on drug-induced pathway between ER stress inducer, tunicamycin and thapsigargin. Using the proteome data during developing iNrich, I found 17 novel translation initiation sites supported both by presence of N-terminal peptides and ribo-seq results in S. cerevisiae, and 26 N-terminal formylated peptides in human cancer cell line. The iNrich facilitated high-throughput N-terminomics and degradomics at low cost using commercially available reagents and apparatus, without requiring arduous procedures.