Discovery of coding regions in the human genome by integrated proteogenomics analysis workflow

Abstract

Proteogenomics enable the discovery of novel peptides (from unannotated genomic protein-coding loci) and single amino acid variant peptides (derived from single-nucleotide polymorphisms and mutations). Increasing the reliability of these identifications is crucial to ensure their usefulness for genome annotation and potential application as neoantigens in cancer immunotherapy. We here present integrated proteogenomics analysis workflow (IPAW), which combines peptide discovery, curation, and validation. IPAW includes the SpectrumAI tool for automated inspection of MS/MS spectra, eliminating false identifications of single-residue substitution peptides. We employ IPAW to analyze two proteomics data sets acquired from A431 cells and five normal human tissues using extended (pH range, 3–10) high-resolution isoelectric focusing (HiRIEF) pre-fractionation and TMT-based peptide quantitation. The IPAW results provide evidence for the translation of pseudogenes, lncRNAs, short ORFs, alternative ORFs, N-terminal extensions, and intronic sequences. Moreover, our quantitative analysis indicates that protein production from certain pseudogenes and lncRNAs is tissue specific.

Proteins encoded by Novel ORFs have increased disorder but can be biochemically regulated and harbour pathogenic mutations

“…With the advent of deep sequencing strategies in both genomics and proteomics fields, we are now discovering nORFs that have remained undiscovered or 'hidden' 1,2,5,6 . These nORFs are pervasive throughout the genome and are observed in both the coding and non coding regions 1,7 . They are variously classified as small ORFs (sORFs) 8,9 which are 1-100 amino acids in length, altORFs 10 , which are proteins in alternate frames to known proteins, Denovogenes 11 or Orphan genes 12 , Pseudogenes 1,13 , and many ncRNAs have been shown to have coding potential [14][15][16][17][18] .…”

Integrated Transcriptomic and Proteomic Analysis of Primary Human Umbilical Vein Endothelial Cells

“…FXR2 and NPLOC4 were identified with an upstream peptide, which was in‐frame with annotated start site (Figure S1, Supporting Information). Recent studies have also reported putative alternate N‐termini for these proteins . For the remaining five proteins, we identified peptides that were direct extensions from annotated protein N‐termini into the 5’‐UTR region which suggests the existence of an alternate N‐terminus or multiple N‐termini.…”

Proteogenomics analysis of non-coding region encoded peptides in normal tissues and five cancer types

“…A few representative tissue-specific pseudogenes/ lncRNAs are listed in Table 1. We detected two previously reported tissue specific non-coding gene translation, testis specific TATDN2P1 (TatD DNase domain containing 2 pseudogene 1, supported by two unique peptides) and placenta specific lncRNA lnc-CACNG8-28:1 (supported by eight unique peptides) 4 . In addition, some more novel tissue specific non-coding gene encoded peptides were discovered in different tissues (supplementary table S2).…”

“…Recently, many mass spectrometry based proteomics studies have identified peptides from non-coding regions of human genome [1][2][3][4][5] .Some peptides are identified from genomic regions in proximity to protein-coding genes, which indicate incorrect exon boundary or a missed exon. Others are identified from currently annotated non-coding sequences include pseudogenes, lncRNAs, protein-coding gene's untranslated region, alternative reading frame or anti-sense strand.…”

“…Here, we analyzed publicly available proteomics data from 926 cancer samples of five cancer types, and 31 different healthy human tissues using previously developed proteogenomics pipeline 4 . With the data, we aim to find out what non-coding sequences including pseudogenes/lncRNAs are actively translated in healthy and tumor tissues and if they exert tissue specific or cancer specific expression.…”