IsobarPTM: A software tool for the quantitative analysis of post-translationally modified proteins☆

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Abstract

The establishment of extremely powerful proteomics platforms able to map thousands of modification sites, e.g. phosphorylations or acetylations, over entire proteomes calls for equally powerful software tools to effectively extract useful and reliable information from such complex datasets. We present a new quantitative PTM analysis platform aimed at processing iTRAQ or Tandem Mass Tags (TMT) labeled peptides. It covers a broad range of needs associated with proper PTM ratio analysis such as PTM localization validation, robust ratio computation and statistical assessment, and navigable user report generation. Isobar ^PTM is made available as an R Bioconductor package and it can be run from the command line by non R specialists.

Biological significance

“IsobarPTM is a new software tool facilitating the quantitative analysis of protein modification regulation streamlining important issues related to PTM localization and statistical modeling. Users are provided with a navigable spreadsheet report, which also annotate already public modification sites.” This article is part of a Special Issue entitled: From Genome to Proteome: Open Innovations.

Graphical abstract

Highlights

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Isobar ^PTM is a new open source software tool to analyze PTM regulation.
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Quantitative iTRAQ and TMT data are supported.
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Whole set of functionalities from PTM size localization to statistical modeling
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Navigable user reports are generated automatically.
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Available as R Bioconductor package

Related collections

Most cited references 27

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Systematic and quantitative assessment of the ubiquitin-modified proteome.

Woong Kim, Eric J. Bennett, Edward L. Huttlin … (2011)

Despite the diverse biological pathways known to be regulated by ubiquitylation, global identification of substrates that are targeted for ubiquitylation has remained a challenge. To globally characterize the human ubiquitin-modified proteome (ubiquitinome), we utilized a monoclonal antibody that recognizes diglycine (diGly)-containing isopeptides following trypsin digestion. We identify ~19,000 diGly-modified lysine residues within ~5000 proteins. Using quantitative proteomics we monitored temporal changes in diGly site abundance in response to both proteasomal and translational inhibition, indicating both a dependence on ongoing translation to observe alterations in site abundance and distinct dynamics of individual modified lysines in response to proteasome inhibition. Further, we demonstrate that quantitative diGly proteomics can be utilized to identify substrates for cullin-RING ubiquitin ligases. Interrogation of the ubiquitinome allows for not only a quantitative assessment of alterations in protein homeostasis fidelity, but also identification of substrates for individual ubiquitin pathway enzymes. Copyright © 2011 Elsevier Inc. All rights reserved.

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Is Open Access

PhosphoSitePlus: a comprehensive resource for investigating the structure and function of experimentally determined post-translational modifications in man and mouse

Peter V. Hornbeck, Jon M. Kornhauser, Sasha Tkachev … (2011)

PhosphoSitePlus (http://www.phosphosite.org) is an open, comprehensive, manually curated and interactive resource for studying experimentally observed post-translational modifications, primarily of human and mouse proteins. It encompasses 1 30 000 non-redundant modification sites, primarily phosphorylation, ubiquitinylation and acetylation. The interface is designed for clarity and ease of navigation. From the home page, users can launch simple or complex searches and browse high-throughput data sets by disease, tissue or cell line. Searches can be restricted by specific treatments, protein types, domains, cellular components, disease, cell types, cell lines, tissue and sequences or motifs. A few clicks of the mouse will take users to substrate pages or protein pages with sites, sequences, domain diagrams and molecular visualization of side-chains known to be modified; to site pages with information about how the modified site relates to the functions of specific proteins and cellular processes and to curated information pages summarizing the details from one record. PyMOL and Chimera scripts that colorize reactive groups on residues that are modified can be downloaded. Features designed to facilitate proteomic analyses include downloads of modification sites, kinase–substrate data sets, sequence logo generators, a Cytoscape plugin and BioPAX download to enable pathway visualization of the kinase–substrate interactions in PhosphoSitePlus®.

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A probability-based approach for high-throughput protein phosphorylation analysis and site localization.

Sean Beausoleil, Judit Villén, Scott A. Gerber … (2006)

Data analysis and interpretation remain major logistical challenges when attempting to identify large numbers of protein phosphorylation sites by nanoscale reverse-phase liquid chromatography/tandem mass spectrometry (LC-MS/MS) (Supplementary Figure 1 online). In this report we address challenges that are often only addressable by laborious manual validation, including data set error, data set sensitivity and phosphorylation site localization. We provide a large-scale phosphorylation data set with a measured error rate as determined by the target-decoy approach, we demonstrate an approach to maximize data set sensitivity by efficiently distracting incorrect peptide spectral matches (PSMs), and we present a probability-based score, the Ascore, that measures the probability of correct phosphorylation site localization based on the presence and intensity of site-determining ions in MS/MS spectra. We applied our methods in a fully automated fashion to nocodazole-arrested HeLa cell lysate where we identified 1,761 nonredundant phosphorylation sites from 491 proteins with a peptide false-positive rate of 1.3%.

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Author and article information

Journal

Journal ID (nlm-ta): J Proteomics

Journal ID (iso-abbrev): J Proteomics

Title: Journal of Proteomics

Publisher: Elsevier

ISSN (Print): 1874-3919

ISSN (Electronic): 1876-7737

Publication date PMC-release: 02 September 2013

Publication date (Print): 02 September 2013

Volume: 90

Issue: 100

Pages: 77-84

Affiliations

CeMM — Research Center for Molecular Medicine of the Austrian Academy of Sciences, Lazarettgasse 14, AKH-BT. 25.3, 1090 Vienna, Austria

Author notes

[* ]Corresponding author. Tel.: + 43 14016070020; fax: + 43 140160970000. jcolinge@ 123456cemm.oeaw.ac.at

Article

Publisher ID: JPROT1331

DOI: 10.1016/j.jprot.2013.02.022

PMC ID: 3759844

PubMed ID: 23470796

SO-VID: e33b7220-829f-4f67-ac04-e67262cd5d8f

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This document may be redistributed and reused, subject to certain conditions.

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