Comprehensive analysis of human protein N-termini enables assessment of various protein forms

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.

N-terminal acetylation and the N-end rule pathway control degradation of the lipid droplet protein PLIN2

“…5). Moreover, global N-terminome analyses reveal that Ala, Ser, or Thr N termini of proteomes are more acetylated than Val or Cys N termini (41,42). Complying with the varying efficacy of Nt-acetylation depending upon the species of Nt-residues, the split-Ub results also demonstrate that TEB4 interacts preferentially with A-PLIN2, S-PLIN2, or T-PLIN2 bearing Nt-acetylation-prone Nt-residues than otherwise identical, but rarely or almost never Nt-acetylated V-PLIN2, C-PLIN2, G-PLIN2, or P-PLIN2 ( Fig.…”

Streamlined selection of cancer antigens for vaccine development through integrative multi-omics and high-content cell imaging

“…The LC-MS/MS analysis was performed as previously described 59 . Dried peptide samples were reconstituted in 10 µL of 0.1% formic acid, and an aliquot containing ~4 μL was injected from a cooled (10 °C) autosampler into a reversed-phase Magic C18aq column (15 cm × 75 μm (packed in-house); Michrom BioResources, Auburn, CA, USA) on an Eksigent nanoLC 2D system at a flow rate of 300 nL/min.…”

Isolation of protein N-terminal peptides by charge-mediated position-selective enrichment using strong cation exchange chromatography

“…The N-terminal peptides of His-containing proteins could be successfully separated from the internal peptides of human and bacterial samples by means of CHAMP-SCX under optimized elution conditions. To validate the applicability of this method to large-scale N-terminal proteomics, we performed triplicate analyses using HEK293T cells, which have been widely used in N-terminal proteomics (17,18). Triplicate SCX fractionations using 10 mM KCl isocratic elution were done for TrypN-digested HEK293T peptides (80 μg each time), and we subjected one-fourth of the isolated peptides to nanoLC/MS/MS in triplicate (9 runs in total).…”

“…Note that 1,600 additional neo-N-terminal peptides were identified when semi-specific cleavage at the N-terminus was allowed in the data processing, although our purpose in this study was not to find novel proteoforms, but to establish a novel approach for N-terminomics. Furthermore, to compare our results with two published N-terminome datasets for HEK293T human cells (17,18), we re-analyzed those datasets under the same conditions without the use of their original customized database or non-specific cleavage. In terms of the contents of acetylated and unmodified protein N-terminal peptides, all three datasets provided identical results, whereas the content of internal peptides as well as the number of unique peptides varied depending on the approach and the sample amount (Supplementary Figure S4).…”

“…Thus, relatively large amounts of samples (~5 to 10 mg) are generally required to increase the identification number of protein N-terminal peptides. This limits the usefulness of these approaches in the case of hard-to-obtain biological samples (17,18). Furthermore, limitations in the efficiency and specificity of the chemical derivatizations compromise the confidence of peptide HEK293T cell culture and protein extraction HEK293T (human embryonic kidney) cells, cultured to 80% confluence in 10-cm diameter dishes, were harvested in lysis buffer containing protease inhibitors (Sigma), 12 mM SDC, 12 mM SLS, 10 mM TCEP, 40 mM CAA in 100 mM Tris buffer (pH 8.5).…”