Identifying specific protein interaction partners using quantitative mass spectrometry and bead proteomes
Laura Trinkle-Mulcahy,Séverine Boulon,Yun Wah Lam,Roby Urcia,François-Michel Boisvert,Franck Vandermoere,Nick A. Morrice,Sam Swift,Ulrich Rothbauer,Heinrich Leonhardt,Angus I. Lamond +10 more
TLDR
A reliable affinity purification strategy to identify specific interactors that combines quantitative SILAC-based mass spectrometry with characterization of common contaminants binding to affinity matrices (bead proteomes) is presented.Abstract:
The identification of interaction partners in protein complexes is a major goal in cell biology. Here we present a reliable affinity purification strategy to identify specific interactors that combines quantitative SILAC-based mass spectrometry with characterization of common contaminants binding to affinity matrices (bead proteomes). This strategy can be applied to affinity purification of either tagged fusion protein complexes or endogenous protein complexes, illustrated here using the well-characterized SMN complex as a model. GFP is used as the tag of choice because it shows minimal nonspecific binding to mammalian cell proteins, can be quantitatively depleted from cell extracts, and allows the integration of biochemical protein interaction data with in vivo measurements using fluorescence microscopy. Proteins binding nonspecifically to the most commonly used affinity matrices were determined using quantitative mass spectrometry, revealing important differences that affect experimental design. These data provide a specificity filter to distinguish specific protein binding partners in both quantitative and nonquantitative pull-down and immunoprecipitation experiments.read more
Citations
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The CRAPome: a contaminant repository for affinity purification–mass spectrometry data
Dattatreya Mellacheruvu,Zachary Wright,Amber L. Couzens,Jean-Philippe Lambert,Nicole St-Denis,Tuo Li,Yana Miteva,Simon Hauri,Mihaela E. Sardiu,Teck Yew Low,Vincentius A. Halim,Vincentius A. Halim,Richard D. Bagshaw,Nina C. Hubner,Abdallah Al-Hakim,Annie Bouchard,Denis Faubert,Damian Fermin,Wade H. Dunham,Marilyn Goudreault,Zhen Yuan Lin,Beatriz Gonzalez Badillo,Tony Pawson,Daniel Durocher,Benoit Coulombe,Ruedi Aebersold,Ruedi Aebersold,Giulio Superti-Furga,Jacques Colinge,Albert J. R. Heck,Hyungwon Choi,Matthias Gstaiger,Shabaz Mohammed,Ileana M. Cristea,Keiryn L. Bennett,Michael P. Washburn,Michael P. Washburn,Brian Raught,Rob M. Ewing,Rob M. Ewing,Anne-Claude Gingras,Alexey I. Nesvizhskii +41 more
TL;DR: The contaminant repository for affinity purification (the CRAPome) is presented and its use for scoring protein-protein interactions is described and aggregating negative controls from multiple AP-MS studies can increase coverage and improve the characterization of background associated with a given experimental protocol.
Journal ArticleDOI
A day in the life of the spliceosome
A. Gregory Matera,Zefeng Wang +1 more
TL;DR: Insights into these questions have been gained by studying the life cycle of spliceosomal snRNAs from their transcription, nuclear export and re-import to their dynamic assembly into thespliceosome.
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Gold nanoparticles of diameter 1.4 nm trigger necrosis by oxidative stress and mitochondrial damage.
Yu Pan,Annika Leifert,David Ruau,Sabine Neuss,Jörg Bornemann,Günter Schmid,Wolfgang Brandau,Ulrich Simon,Willi Jahnen-Dechent +8 more
TL;DR: Gold nanoparticles (AuNPs) are generally considered nontoxic, similar to bulk gold, which is inert and biocompatible, but here, ligand chemistry is a critical parameter determining the degree of cytotoxicity.
Journal ArticleDOI
Mapping intact protein isoforms in discovery mode using top-down proteomics
John C. Tran,Leonid Zamdborg,Dorothy R. Ahlf,Dorothy R. Ahlf,Ji Eun Lee,Ji Eun Lee,Adam D. Catherman,Adam D. Catherman,Kenneth R. Durbin,Kenneth R. Durbin,Jeremiah D. Tipton,Adaikkalam Vellaichamy,Adaikkalam Vellaichamy,John F. Kellie,John F. Kellie,Mingxi Li,Mingxi Li,Cong Wu,Steve M. M. Sweet,Steve M. M. Sweet,Bryan P. Early,Bryan P. Early,Nertila Siuti,Nertila Siuti,Richard D. LeDuc,Philip D. Compton,Paul M. Thomas,Paul M. Thomas,Neil L. Kelleher,Neil L. Kelleher +29 more
TL;DR: Identification of 1,043 gene products from human cells that are dispersed into more than 3,000 protein species created by post-translational modification, RNA splicing and proteolysis is shown, using a new four-dimensional separation system.
Journal ArticleDOI
Mass spectrometry–based proteomics in cell biology
Tobias C. Walther,Matthias Mann +1 more
TL;DR: Rapid advances in all areas of the proteomic workflow, including sample preparation, MS, and computational analysis, should make the technology more easily available to a broad community and turn it into a staple methodology for cell biologists.
References
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Shao En Ong,Blagoy Blagoev,Irina Kratchmarova,Dan B. Kristensen,Hanno Steen,Akhilesh Pandey,Matthias Mann +6 more
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Journal ArticleDOI
A proteomics strategy to elucidate functional protein-protein interactions applied to EGF signaling
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