Abstract
Various forms of protein (proteoforms) are generated by genetic variations, alternative splicing, alternative translation initiation, co- or post-translational modification and proteolysis. Different proteoforms are in part discovered by characterizing their N-terminal sequences. Here, we introduce an N-terminal-peptide-enrichment method, Nrich. Filter-aided negative selection formed the basis for the use of two N-blocking reagents and two endoproteases in this method. We identified 6,525 acetylated (or partially acetylated) and 6,570 free protein N-termini arising from 5,727 proteins in HEK293T human cells. The protein N-termini included translation initiation sites annotated in the UniProtKB database, putative alternative translational initiation sites, and N-terminal sites exposed after signal/transit/pro-peptide removal or unknown processing, revealing various proteoforms in cells. In addition, 46 novel protein N-termini were identified in 5′ untranslated region (UTR) sequence with pseudo start codons. Our data showing the observation of N-terminal sequences of mature proteins constitutes a useful resource that may provide information for a better understanding of various proteoforms in cells.
Highlights
The diversity of protein from a single gene is generated by several mechanisms, including co- or post-translational modification, alternative splicing and alternative translation initiation
In order to characterize the status of proteins in cells, we performed “deep-down” N-terminal peptide enrichment (Nrich) in HEK293T cell line based on a negative selection method (Fig. 1, Methods)
N-terminal peptides enrichment on the filter (Nrich) consisted of three major experimental steps: 1) the first step was to distinguish between endogenous Nα -acetylated and endogenous free N-termini
Summary
The diversity of protein from a single gene is generated by several mechanisms, including co- or post-translational modification, alternative splicing and alternative translation initiation. The approaches for identification of the N-termini of proteins are based on bottom-up proteomics and have been developed as positive or negative selection methods by which protein N-terminal peptides are isolated from other digested internal peptides. These approaches have recently been comprehensively reviewed[8, 9]. There was a report that found ~7,900 protein N-termini in human B cells by TAILS method without peptide prefractionation[18] These approaches for the identification of protein N-termini have been successfully applied to many samples measuring protein turnover, determining translation initiation sites and confirming protein degradation, information of N-terminus for many proteins is still missing. The identification of N-terminome might provide a better understanding of various proteoforms in cells
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