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

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.Proteogenomics enables the discovery of protein coding regions and disease-relevant mutations but their verification remains challenging. Here, the authors combine peptide discovery, curation and validation in an integrated proteogenomics workflow, robustly identifying unknown coding regions and mutations.

• Publication year

  2018

• Venue

  Nature Communications

• Publication date

  2018-03-02

• Fields of study

  Biology, Medicine

• Identifiers
  DOI 10.1038/s41467-018-03311-yPMID 29500430PMCID 5834625
• External record

  Open on Semantic Scholar

• Source metadata

  Semantic Scholar, PubMed

• No claims are published for this paper.

• No concepts are published for this paper.

• Integrated Proteogenomic Characterization of Human High-Grade Serous Ovarian Cancer.

  2025cited by this paper
• Sequence Analysis

  2020cited by this paper
• Exploiting non-canonical translation to identify new targets for T cell-based cancer immunotherapy

  2018cited by this paper
• Applications of Immunogenomics to Cancer.

  2017cited by this paper
• Evaluating the effect of database inflation in proteogenomic search on sensitive and reliable peptide identification

  2016cited by this paper
• Integrated Proteogenomic Characterization of Human High-Grade Serous Ovarian Cancer.

  2016cited by this paper
• JUMPg: An Integrative Proteogenomics Pipeline Identifying Unannotated Proteins in Human Brain and Cancer Cells.

  2016cited by this paper
• Translational control by 5′-untranslated regions of eukaryotic mRNAs

  2016cited by this paper
• Proteogenomics connects somatic mutations to signaling in breast cancer

  2016cited by this paper
• Global proteogenomic analysis of human MHC class I-associated peptides derived from non-canonical reading frames

  2016cited by this paper
• Improving GENCODE reference gene annotation using a high-stringency proteogenomics workflow

  2016cited by this paper
• COSMIC: somatic cancer genetics at high-resolution

  2016cited by this paper
• Death of a dogma: eukaryotic mRNAs can code for more than one protein

  2015cited by this paper
• Many lncRNAs, 5’UTRs, and pseudogenes are translated and some are likely to express functional proteins

  2015cited by this paper
• Tissue-based map of the human proteome

  2015cited by this paper
• PGTools: A Software Suite for Proteogenomic Data Analysis and Visualization.

  2015cited by this paper
• A note on the false discovery rate of novel peptides in proteogenomics

  2015cited by this paper
• Sambamba: fast processing of NGS alignment formats

  2015cited by this paper
• Found in translation: functions and evolution of a recently discovered alternative proteome.

  2015cited by this paper
• Devising a Consensus Framework for Validation of Novel Human Coding Loci.

  2015cited by this paper
• A Scalable Approach for Protein False Discovery Rate Estimation in Large Proteomic Data Sets

  2015cited by this paper
• The Pan-Cancer Analysis of Pseudogene Expression Reveals Biologically and Clinically Relevant Tumour Subtypes

  2014cited by this paper
• Proteogenomic characterization of human colon and rectal cancer

  2014cited by this paper
• A promoter-level mammalian expression atlas

  2014cited by this paper
• A draft map of the human proteome

  2014cited by this paper
• Mass-spectrometry-based draft of the human proteome

  2014cited by this paper
• Proteogenomics: concepts, applications and computational strategies

  2014cited by this paper
• Universal database search tool for proteomics

  2014cited by this paper
• Analyzing the First Drafts of the Human Proteome

  2014cited by this paper
• Using Galaxy-P to leverage RNA-Seq for the discovery of novel protein variations

  2014cited by this paper
• Annotating N Termini for the Human Proteome Project: N Termini and Nα-Acetylation Status Differentiate Stable Cleaved Protein Species from Degradation Remnants in the Human Erythrocyte Proteome

  2014cited by this paper
• Emerging evidence for functional peptides encoded by short open reading frames

  2014cited by this paper
• Proteomics techniques for the detection of translated pseudogenes.

  2014cited by this paper
• Reprogramming activity of NANOGP8, a NANOG family member widely expressed in cancer

  2014cited by this paper
• Onco-proteogenomics: cancer proteomics joins forces with genomics

  2014cited by this paper
• customProDB: an R package to generate customized protein databases from RNA-Seq data for proteomics search

  2013cited by this paper
• Ribosome profiling provides evidence that large noncoding RNAs do not encode proteins.

  2013cited by this paper
• HiRIEF LC-MS enables deep proteome coverage and unbiased proteogenomics

  2013cited by this paper
• STAR: ultrafast universal RNA-seq aligner

  2013cited by this paper
• Deep Proteome Coverage Based on Ribosome Profiling Aids Mass Spectrometry-based Protein and Peptide Discovery and Provides Evidence of Alternative Translation Products and Near-cognate Translation Initiation Events*

  2013cited by this paper
• Discovery and Mass Spectrometric Analysis of Novel Splice-junction Peptides Using RNA-Seq*

  2013cited by this paper
• Direct Detection of Alternative Open Reading Frames Translation Products in Human Significantly Expands the Proteome

  2013cited by this paper
• Computer aided manual validation of mass spectrometry-based proteomic data.

  2013cited by this paper
• LNCipedia: a database for annotated human lncRNA transcript sequences and structures

  2012cited by this paper
• GENCODE: The reference human genome annotation for The ENCODE Project

  2012cited by this paper
• MSnbase-an R/Bioconductor package for isobaric tagged mass spectrometry data visualization, processing and quantitation

  2012cited by this paper
• A Cross-platform Toolkit for Mass Spectrometry and Proteomics

  2012cited by this paper
• Peptidomic discovery of short open reading frame-encoded peptides in human cells

  2012cited by this paper
• Pseudogenes: Newly Discovered Players in Human Cancer

  2012cited by this paper
• Comprehensive analysis of the genome transcriptome and proteome landscapes of three tumor cell lines

  2012cited by this paper
• Technical updates to basic proteins focalization using IPG strips

  2012cited by this paper
• Genome-wide search for novel human uORFs and N-terminal protein extensions using ribosomal footprinting

  2012cited by this paper
• The GENCODE pseudogene resource

  2012cited by this paper
• Expressed pseudogenes in the transcriptional landscape of human cancers.

  2012cited by this paper
• Long noncoding RNAs are rarely translated in two human cell lines

  2012cited by this paper
• PhyloCSF: a comparative genomics method to distinguish protein coding and non-coding regions

  2011cited by this paper
• A Bioinformatics Workflow for Variant Peptide Detection in Shotgun Proteomics*

  2011cited by this paper
• Detection of nonneutral substitution rates on mammalian phylogenies.

  2010cited by this paper
• Fast and accurate long-read alignment with Burrows–Wheeler transform

  2010cited by this paper
• CanProVar: a human cancer proteome variation database

  2010cited by this paper
• Statistical distribution of amino acid sequences: a proof of Darwinian evolution

  2010cited by this paper
• Genome-Wide Analysis in Vivo of Translation with Nucleotide Resolution Using Ribosome Profiling

  2009cited by this paper
• The Sequence Alignment/Map format and SAMtools

  2009cited by this paper
• Universal sample preparation method for proteome analysis

  2009cited by this paper
• Whole genome searching with shotgun proteomic data: applications for genome annotation.

  2008cited by this paper
• Locating proteins in the cell using TargetP, SignalP and related tools

  2007cited by this paper
• Semi-supervised learning for peptide identification from shotgun proteomics datasets

  2007cited by this paper
• Locating proteins in the cell using TargetP, SignalP and related tools

  2007cited by this paper
• Evolutionarily conserved elements in vertebrate, insect, worm, and yeast genomes.

  2005cited by this paper
• Theoretical and practical advances in genome halving

  2004cited by this paper
• BLAT--the BLAST-like alignment tool.

  2002cited by this paper
• Gapped BLAST and PSI-BLAST: a new generation of protein database search programs.

  1997cited by this paper

• A label-free microLC-SWATH-MS methodology with immunoaffinity depletion of highly abundant serum proteins for quantitative proteomic comparison of fresh-frozen human normal breast tissue and tumor clinical specimens.

  2026cites this paper
• Harnessing Noncanonical Proteins for Next‐Generation Drug Discovery and Diagnosis

  2025cites this paper
• Programmed Cell Death Protein 10 (PDCD10) Regulates Vesicle Trafficking and Contributes to the Progression of Clear Cell Renal Cell Carcinoma

  2025cites this paper
• The ‘non-conventional’ peptidome: A new layer in plant regulatory mechanisms

  2025cites this paper
• Proteogenomics-enabled discovery of novel small open reading frame (sORF)-encoded polypeptides in human and mouse tissues.

  2025cites this paper
• The degradation of extended protein isoforms points to a misfiring translation initiation process

  2025influential citation
• Assessing the Performance of Mass Spectrometry Search Strategies in Identifying Translational Errors Using PDX Proteomics Data

  2025cites this paper
• Intrinsic and non-cell autonomous roles for a neurodevelopmental syndrome-linked transcription factor

  2025cites this paper
• Functional-proteomics-based investigation of the cellular response to farnesyltransferase inhibition in lung cancer

  2025cites this paper
• A large-scale sORF screen identifies putative microproteins involved in cancer cell fitness

  2025cites this paper
• Proteogenomic Approaches for Diseasome Studies.

  2025cites this paper
• Introduction to Integrated Proteogenomic Pipeline for Dealing with Pathogenic Missense SNPs.

  2025cites this paper
• Proteogenomic Profiling of Treatment-Naïve Metastatic Malignant Melanoma

  2025cites this paper
• Suppression of pseudogene MT2P1 transcription induced by E2F7 inhibits hepatocellular carcinoma cell proliferation and facilitates apoptosis via preserving its parental gene

  2025cites this paper
• Enhanced Discovery of Alternative Proteins (AltProts) in Mouse Cardiac Development Using Data-Independent Acquisition (DIA) Proteomics

  2025cites this paper
• sOCP: a framework predicting smORF coding potential based on TIS and in-frame features and effectively applied in the human genome

  2024cites this paper
• High‐throughput molecular assays for inclusion in personalised oncology trials – State‐of‐the‐art and beyond

  2024cites this paper
• Harnessing the power of proteomics in precision diabetes medicine

  2024cites this paper
• Chemoproteogenomic stratification of the missense variant cysteinome

  2024cites this paper
• Proteomics Can Rise to the Challenge of Pseudogenes’ Coding Nature

  2024cites this paper
• A deep audit of the PeptideAtlas database uncovers evidence for unannotated coding genes and aberrant translation

  2024cites this paper
• Proteogenomic analysis reveals non-small cell lung cancer subtypes predicting chromosome instability, and tumor microenvironment

  2024cites this paper
• The rapid degradation of translated upstream regions points to an inefficient translation initiation process

  2024influential citation
• The dual‐coding gene SLC35A4 protects against oxidative stress

  2024cites this paper
• Detection of host cell microprotein impurities in antibody drug products

  2024cites this paper
• Development of novel peptides filtering algorithm based on peptide information

  2024cites this paper
• ncRNA Coding Potential Prediction Using BiLSTM and Transformer Encoder-Based Model

  2024cites this paper
• Identification of Novel Bacterial Microproteins Encoded by Small Open Reading Frames Using a Computational Proteogenomics Workflow.

  2024cites this paper
• Rp3: Ribosome profiling-assisted proteogenomics improves coverage and confidence during microprotein discovery

  2024cites this paper
• Evidence for widespread translation of 5′ untranslated regions

  2024influential citation
• Proteogenomics analysis of human tissues using pangenomes

  2024influential citation
• A Tale of Three Proteomes: Visualizing Protein and Transcript Abundance Relationships in the Breast Cancer Proteome Portal.

  2023cites this paper
• Variant biomarker discovery using mass spectrometry-based proteogenomics

  2023cites this paper
• PABPN1 prevents the nuclear export of an unspliced RNA with a constitutive transport element and controls human gene expression via intron retention

  2023cites this paper
• OpenCustomDB: Integration of Unannotated Open Reading Frames and Genetic Variants to Generate More Comprehensive Customized Protein Databases

  2023cites this paper
• Annotation of the non-canonical translatome reveals that CHO cell microproteins are a new class of mAb drug product impurity

  2023cites this paper
• PgxSAVy: A tool for comprehensive evaluation of variant peptide quality in proteogenomics – catching the (un)usual suspects

  2023cites this paper
• Integration of proteomics in the molecular tumor board

  2023cites this paper
• Proteogenomics 101: a primer on database search strategies

  2023cites this paper
• A Program for Predicting the Retention Time of Peptides with Post-Translational Modifications

  2023cites this paper
• Protein-coding potential of non-canonical open reading frames in human transcriptome.

  2023cites this paper
• Histopathology-assisted proteogenomics provides foundations for stratification of melanoma metastases

  2023cites this paper
• The Integration of Proteogenomics and Ribosome Profiling Circumvents Key Limitations to Increase the Coverage and Confidence of Novel Microproteins

  2023cites this paper
• Proteogenomics in Cancer: Then and Now.

  2023cites this paper
• Multi-omic stratification of the missense variant cysteinome

  2023cites this paper
• Quality control of variant peptides identified through proteogenomics- catching the (un)usual suspects

  2023cites this paper
• A large-scale sORF screen identifies putative microproteins and provides insights into their interaction partners, localisation and function

  2023cites this paper
• Quality control and annotation of variant peptides identified through Proteogenomics

  2023cites this paper
• The Breast Cancer Proteome and Precision Oncology.

  2023cites this paper
• Large-scale proteogenomics characterization of microproteins in Mycobacterium tuberculosis

  2023cites this paper
• Microproteins: Overlooked regulators of physiology and disease

  2023cites this paper
• Proteogenomic analysis of lung adenocarcinoma reveals tumor heterogeneity, survival determinants, and therapeutically relevant pathways

  2022cites this paper
• Identification of Novel Genes and Proteoforms in Angiostrongylus costaricensis through a Proteogenomic Approach

  2022cites this paper
• Pseudogene UBE2MP1 derived transcript enhances in vitro cell proliferation and apoptosis resistance of hepatocellular carcinoma cells through miR-145-5p/RGS3 axis

  2022cites this paper
• The proteomic landscape of glioblastoma recurrence reveals novel and targetable immunoregulatory drivers

  2022cites this paper
• Proteogenomic Analysis of Breast Cancer Transcriptomic and Proteomic Data, Using De Novo Transcript Assembly: Genome-Wide Identification of Novel Peptides and Clinical Implications

  2022cites this paper
• OpenVar: functional annotation of variants in non-canonical open reading frames

  2022cites this paper
• Limited Evidence for Protein Products of Noncoding Transcripts in the HEK293T Cellular Cytosol

  2022cites this paper
• Novel insights into noncanonical open reading frames in cancer.

  2022cites this paper
• Novel markers for liquid biopsies in cancer management: Circulating platelets and extracellular vesicles.

  2022cites this paper
• Small open reading frames in plant research: from prediction to functional characterization

  2022cites this paper
• False discovery rate: the Achilles' heel of proteogenomics

  2022cites this paper
• Multiaspect Examinations of Possible Alternative Mappings of Identified Variant Peptides: A Case Study on the HEK293 Cell Line

  2022cites this paper
• Proteomics‐driven identification of short open reading frame‐encoded peptides

  2022cites this paper
• Proteomics approaches to characterize the immune responses in cancer.

  2022cites this paper
• Short open reading frames (sORFs) and microproteins: an update on their identification and validation measures

  2022cites this paper
• Glioblastoma stem cells express non‐canonical proteins and exclusive mesenchymal‐like or non‐mesenchymal‐like protein signatures

  2022cites this paper
• The Immunological Conundrum of Endogenous Retroelements

  2022cites this paper
• Proteogenomic analysis of acute myeloid leukemia associates relapsed disease with reprogrammed energy metabolism both in adults and children

  2022cites this paper
• Pseudogene SNRPFP1 derived long non-coding RNA facilitates hepatocellular carcinoma progress in vitro by sponging tumor-suppressive miR-126-5p

  2022cites this paper
• Identification of multiple TAR DNA binding protein retropseudogene lineages during the evolution of primates

  2021cites this paper
• A natural nanozyme in life is found: the iron core within ferritin shows superoxide dismutase catalytic activity

  2021cites this paper
• Pan-cancer analysis of transcripts encoding novel open-reading frames (nORFs) and their potential biological functions

  2021cites this paper
• In silico identification of novel open reading frames in Plasmodium falciparum oocyte and salivary gland sporozoites using proteogenomics framework

  2021cites this paper
• Revisiting sORFs: overcoming challenges to identify and characterize functional microproteins

  2021cites this paper
• The transcriptome‐wide landscape of molecular subtype‐specific mRNA expression profiles in acute myeloid leukemia

  2021cites this paper
• LncMachine: a machine learning algorithm for long noncoding RNA annotation in plants

  2021cites this paper
• Increased expression of peptides from non-coding genes in cancer proteomics datasets suggests potential tumor neoantigens

  2021cites this paper
• Omics Sciences for Systems Biology in Alzheimer's Disease: State-of-the-Art of the Evidence.

  2021cites this paper
• CREB-mediated transcriptional activation of NRMT1 drives muscle differentiation

  2021cites this paper
• Generation of ENSEMBL-based proteogenomics databases boosts the identification of non-canonical peptides

  2021cites this paper
• A community-driven roadmap to advance research on translated open reading frames detected by Ribo-seq

  2021cites this paper
• Kcnj16 (Kir5.1) Gene Ablation Causes Subfertility and Increases the Prevalence of Morphologically Abnormal Spermatozoa

  2021cites this paper
• Application of Pool-seq for variation detection and proteogenomic database creation in β-hemolytic streptococci

  2021cites this paper
• Proteogenomics Integrating Novel Junction Peptide Identification Strategy Discovers Three Novel Protein Isoforms of Human NHSL1 and EEF1B2.

  2021cites this paper
• Protein-gene Expression Nexus: Comprehensive characterization of human cancer cell lines with proteogenomic analysis

  2021cites this paper
• Peptidomics and proteogenomics: background, challenges and future needs

  2021cites this paper
• The role of proteomics in the multiplexed analysis of gene alterations in human cancer

  2021cites this paper
• The Combiome Hypothesis: Selecting Optimal Treatment for Cancer Patients.

  2021cites this paper
• Small Protein Enrichment Improves Proteomics Detection of sORF Encoded Polypeptides

  2021cites this paper
• Psoriasis to Psoriatic Arthritis: The Application of Proteomics Technologies

  2021cites this paper
• Bioinformatics analysis of NSCLC multi-omics data

  2021cites this paper
• Proteogenomics of non-small cell lung cancer reveals molecular subtypes associated with specific therapeutic targets and immune evasion mechanisms

  2021cites this paper
• Mass spectrometry-based proteomic analysis of NSCLC tumor and biopsy samples

  2021cites this paper
• The Small Open Reading Frame‐Encoded Peptides: Advances in Methodologies and Functional Studies

  2021cites this paper
• Limited, but potentially functional translation of non-coding transcripts in the HEK293T cellular cytosol

  2021cites this paper
• Pan-cancer analysis of transcripts encoding novel open-reading frames (nORFs) and their potential biological functions

  2021cites this paper
• Increased expression of peptides from non-coding genes in cancer proteomics datasets suggests potential tumor neoantigens

  2021cites this paper
• Systemic vitamin intake impacting tissue proteomes

  2020cites this paper
• Methods for Proteogenomics Data Analysis, Challenges, and Scalability Bottlenecks: A Survey

  2020influential citation