A New in Vivo Cross-linking Mass Spectrometry Platform to Define Protein–Protein Interactions in Living Cells*

Protein–protein interactions (PPIs) are fundamental to the structure and function of protein complexes. Resolving the physical contacts between proteins as they occur in cells is critical to uncovering the molecular details underlying various cellular activities. To advance the study of PPIs in living cells, we have developed a new in vivo cross-linking mass spectrometry platform that couples a novel membrane-permeable, enrichable, and MS-cleavable cross-linker with multistage tandem mass spectrometry. This strategy permits the effective capture, enrichment, and identification of in vivo cross-linked products from mammalian cells and thus enables the determination of protein interaction interfaces. The utility of the developed method has been demonstrated by profiling PPIs in mammalian cells at the proteome scale and the targeted protein complex level. Our work represents a general approach for studying in vivo PPIs and provides a solid foundation for future studies toward the complete mapping of PPI networks in living systems.

[1]  Juergen Kast,et al.  Identification of protein‐protein interactions using in vivo cross‐linking and mass spectrometry , 2004, Proteomics.

[2]  E. Kiss-Toth,et al.  Advanced technologies for studies on protein interactomes. , 2008, Advances in biochemical engineering/biotechnology.

[3]  Brad T. Sherman,et al.  Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources , 2008, Nature Protocols.

[4]  Andrea Sinz,et al.  Cleavable cross-linker for protein structure analysis: reliable identification of cross-linking products by tandem MS. , 2010, Analytical chemistry.

[5]  Ruedi Aebersold,et al.  Corrigendum: Identification of cross-linked peptides from large sequence databases , 2008 .

[6]  J. Ranish,et al.  An Integrated Chemical Cross-linking and Mass Spectrometry Approach to Study Protein Complex Architecture and Function* , 2011, Molecular & Cellular Proteomics.

[7]  Pierre Baldi,et al.  Mapping the Structural Topology of the Yeast 19S Proteasomal Regulatory Particle Using Chemical Cross-linking and Probabilistic Modeling* , 2012, Molecular & Cellular Proteomics.

[8]  P. Cramer,et al.  Architecture of the RNA polymerase II–TFIIF complex revealed by cross-linking and mass spectrometry , 2010, EMBO Journal.

[9]  R. Aebersold,et al.  Structural Probing of a Protein Phosphatase 2A Network by Chemical Cross-Linking and Mass Spectrometry , 2012, Science.

[10]  G. Anderson,et al.  In vivo identification of the outer membrane protein OmcA-MtrC interaction network in Shewanella oneidensis MR-1 cells using novel hydrophobic chemical cross-linkers. , 2008, Journal of proteome research.

[11]  R. Aebersold,et al.  Molecular architecture of the 26S proteasome holocomplex determined by an integrative approach , 2012, Proceedings of the National Academy of Sciences.

[12]  James E Bruce,et al.  Mass spectrometry identifiable cross-linking strategy for studying protein-protein interactions. , 2005, Analytical chemistry.

[13]  Robyn M. Kaake,et al.  Selective enrichment and identification of azide-tagged cross-linked peptides using chemical ligation and mass spectrometry , 2010, Journal of the American Society for Mass Spectrometry.

[14]  Lan Huang,et al.  Developing New Isotope-Coded Mass Spectrometry-Cleavable Cross-Linkers for Elucidating Protein Structures , 2014, Analytical chemistry.

[15]  Andrea Sinz,et al.  Chemical cross-linking and mass spectrometry to map three-dimensional protein structures and protein-protein interactions. , 2006, Mass spectrometry reviews.

[16]  Christoph H. Borchers,et al.  ICC-CLASS: isotopically-coded cleavable crosslinking analysis software suite , 2009, BMC Bioinformatics.

[17]  Alma L Burlingame,et al.  Isotope-coded and affinity-tagged cross-linking (ICATXL): an efficient strategy to probe protein interaction surfaces. , 2006, Journal of the American Chemical Society.

[18]  Chunxiang Zheng,et al.  Protein Interactions, Post-translational Modifications and Topologies in Human Cells* , 2013, Molecular & Cellular Proteomics.

[19]  Friedrich Förster,et al.  Near-atomic resolution structural model of the yeast 26S proteasome , 2012, Proceedings of the National Academy of Sciences.

[20]  Lan Huang,et al.  An Integrated Mass Spectrometry-based Proteomic Approach , 2006, Molecular & Cellular Proteomics.

[21]  Tony Pawson,et al.  Mapping differential interactomes by affinity purification coupled with data independent mass spectrometry acquisition , 2013, Nature Methods.

[22]  Vishal R. Patel,et al.  Mapping the Protein Interaction Network of the Human COP9 Signalosome Complex Using a Label-free QTAX Strategy* , 2012, Molecular & Cellular Proteomics.

[23]  Ruedi Aebersold,et al.  Identification of cross-linked peptides from large sequence databases , 2008, Nature Methods.

[24]  Christoph H Borchers,et al.  BiPS, a Photocleavable, Isotopically Coded, Fluorescent Cross-linker for Structural Proteomics * , 2009, Molecular & Cellular Proteomics.

[25]  Carolyn R. Bertozzi,et al.  Reactivity of Biarylazacyclooctynones in Copper-Free Click Chemistry , 2012, Journal of the American Chemical Society.

[26]  C. Bertozzi,et al.  Rapid Cu-Free Click Chemistry with Readily Synthesized Biarylazacyclooctynones , 2010, Journal of the American Chemical Society.

[27]  Daniel F Tardiff,et al.  Protein characterization of Saccharomyces cerevisiae RNA polymerase II after in vivo cross-linking , 2007, Proceedings of the National Academy of Sciences.

[28]  Ruedi Aebersold,et al.  Architecture of the large subunit of the mammalian mitochondrial ribosome , 2013, Nature.

[29]  J. Matthews,et al.  Protein-protein interactions in human disease. , 2005, Current opinion in structural biology.

[30]  Gabriel C. Lander,et al.  Complete subunit architecture of the proteasome regulatory particle , 2011, Nature.

[31]  R. Aebersold,et al.  Analysis of protein complexes using mass spectrometry , 2007, Nature Reviews Molecular Cell Biology.

[32]  R. Aebersold,et al.  Probing Native Protein Structures by Chemical Cross-linking, Mass Spectrometry, and Bioinformatics , 2010, Molecular & Cellular Proteomics.

[33]  S. Gygi,et al.  Defining the Human Deubiquitinating Enzyme Interaction Landscape , 2009, Cell.

[34]  C. D. de Koster,et al.  Selective enrichment of azide-containing peptides from complex mixtures. , 2009, Journal of proteome research.

[35]  Friedrich Förster,et al.  False discovery rate estimation for cross-linked peptides identified by mass spectrometry , 2012, Nature Methods.

[36]  Andreas Bracher,et al.  The molecular architecture of the eukaryotic chaperonin TRiC/CCT. , 2012, Structure.

[37]  Zachary A. Szpiech,et al.  High-resolution network biology: connecting sequence with function , 2013, Nature Reviews Genetics.

[38]  M. Dong,et al.  Identification of cross-linked peptides from complex samples , 2012, Nature Methods.

[39]  Yali Lu,et al.  Ionic reagent for controlling the gas-phase fragmentation reactions of cross-linked peptides. , 2008, Analytical chemistry.

[40]  Tony Pawson,et al.  Temporal regulation of EGF signaling networks by the scaffold protein Shc1 , 2013, Nature.

[41]  Pierre Baldi,et al.  A Tandem Affinity Tag for Two-step Purification under Fully Denaturing Conditions , 2006, Molecular & Cellular Proteomics.

[42]  Natasa Przulj,et al.  Characterization of cell cycle specific protein interaction networks of the yeast 26S proteasome complex by the QTAX strategy. , 2010, Journal of proteome research.

[43]  David R Goodlett,et al.  xComb: a cross-linked peptide database approach to protein-protein interaction analysis. , 2010, Journal of proteome research.

[44]  C. D. de Koster,et al.  An Aptly Positioned Azido Group in the Spacer of a Protein Cross‐Linker for Facile Mapping of Lysines in Close Proximity , 2007, Chembiochem : a European journal of chemical biology.

[45]  Arlo Z. Randall,et al.  Development of a Novel Cross-linking Strategy for Fast and Accurate Identification of Cross-linked Peptides of Protein Complexes* , 2010, Molecular & Cellular Proteomics.

[46]  R. Aebersold,et al.  Chemical cross-linking/mass spectrometry targeting acidic residues in proteins and protein complexes , 2014, Proceedings of the National Academy of Sciences.

[47]  Ludovic C. Gillet,et al.  Quantifying protein interaction dynamics by SWATH mass spectrometry: application to the 14-3-3 system , 2013, Nature Methods.

[48]  Robert J. Chalkley,et al.  Matching Cross-linked Peptide Spectra: Only as Good as the Worse Identification* , 2013, Molecular & Cellular Proteomics.

[49]  Ronald J. Moore,et al.  Identification of cross-linked peptides after click-based enrichment using sequential collision-induced dissociation and electron transfer dissociation tandem mass spectrometry. , 2009, Analytical chemistry.

[50]  T. Shea,et al.  Regulation of the transition from vimentin to neurofilaments during neuronal differentiation. , 2003, Cell motility and the cytoskeleton.

[51]  J. Kast,et al.  Utility of formaldehyde cross-linking and mass spectrometry in the study of protein-protein interactions. , 2008, Journal of mass spectrometry : JMS.

[52]  Anne-Claude Gavin,et al.  Recent advances in charting protein-protein interaction: mass spectrometry-based approaches. , 2011, Current opinion in biotechnology.

[53]  F. Cohen,et al.  Time-controlled transcardiac perfusion cross-linking for the study of protein interactions in complex tissues , 2004, Nature Biotechnology.

[54]  Tijana Milenkovic,et al.  Characterization of the proteasome interaction network using a QTAX-based tag-team strategy and protein interaction network analysis , 2008, Proceedings of the National Academy of Sciences.