Title: A Label-free Mass Spectrometry Method to Predict Endogenous Protein Complex Composition*
Abstract: Information on the composition of protein complexes can accelerate mechanistic analyses of cellular systems. Protein complex composition identifies genes that function together and provides clues about regulation within and between cellular pathways. Cytosolic protein complexes control metabolic flux, signal transduction, protein abundance, and the activities of cytoskeletal and endomembrane systems. It has been estimated that one third of all cytosolic proteins in leaves exist in an oligomeric state, yet the composition of nearly all remain unknown. Subunits of stable protein complexes copurify, and combinations of mass-spectrometry-based protein correlation profiling and bioinformatic analyses have been used to predict protein complex subunits. Because of uncertainty regarding the power or availability of bioinformatic data to inform protein complex predictions across diverse species, it would be highly advantageous to predict composition based on elution profile data alone. Here we describe a mass spectrometry-based protein correlation profiling approach to predict the composition of hundreds of protein complexes based on biochemical data. Extracts were obtained from an intact organ and separated in parallel by size and charge under nondenaturing conditions. More than 1000 proteins with reproducible elution profiles across all replicates were subjected to clustering analyses. The resulting dendrograms were used to predict the composition of known and novel protein complexes, including many that are likely to assemble through self-interaction. An array of validation experiments demonstrated that this new method can drive protein complex discovery, guide hypothesis testing, and enable systems-level analyses of protein complex dynamics in any organism with a sequenced genome. Information on the composition of protein complexes can accelerate mechanistic analyses of cellular systems. Protein complex composition identifies genes that function together and provides clues about regulation within and between cellular pathways. Cytosolic protein complexes control metabolic flux, signal transduction, protein abundance, and the activities of cytoskeletal and endomembrane systems. It has been estimated that one third of all cytosolic proteins in leaves exist in an oligomeric state, yet the composition of nearly all remain unknown. Subunits of stable protein complexes copurify, and combinations of mass-spectrometry-based protein correlation profiling and bioinformatic analyses have been used to predict protein complex subunits. Because of uncertainty regarding the power or availability of bioinformatic data to inform protein complex predictions across diverse species, it would be highly advantageous to predict composition based on elution profile data alone. Here we describe a mass spectrometry-based protein correlation profiling approach to predict the composition of hundreds of protein complexes based on biochemical data. Extracts were obtained from an intact organ and separated in parallel by size and charge under nondenaturing conditions. More than 1000 proteins with reproducible elution profiles across all replicates were subjected to clustering analyses. The resulting dendrograms were used to predict the composition of known and novel protein complexes, including many that are likely to assemble through self-interaction. An array of validation experiments demonstrated that this new method can drive protein complex discovery, guide hypothesis testing, and enable systems-level analyses of protein complex dynamics in any organism with a sequenced genome. There are important roles for "omics" technologies to generate systems level data to inform strategies for trait engineering (1Langridge P. Fleury D. Making the most of 'omics' for crop breeding.Trends Biotechnol. 2011; 29: 33-40Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 2Eldakak M. Milad S.I.M. Nawar A.I. Rohila J.S. Proteomics: a biotechnology tool for crop improvement.Frontiers Plant Sci. 2013; 4: 1-12Crossref PubMed Scopus (68) Google Scholar). Information about protein oligomerization is some of the most valuable biological data that can provide insight into the control of metabolic pathways and cellular systems (3Srere Pa Macromolecular interactions: Tracing the roots.Trends Biochem. 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Native complexes can be isolated and identified with antibodies and coimmunoprecipitation (CoIP) 1The abbreviations used are:CoIP-MScoimmunoprecipitation-mass spectrometryIEXion exchange chromatographyMappapparent mass measured by size exclusion chromatographyMcalccalculated mass of a predicted protein complex based on the proteins in clusterMmonocalculated monomer mass for a proteinP200200k × g pellet containing the microsomal fractionRappratio of the apparent mass to the calculated mass of the monomerS200soluble proteins after differential centrifugationSDS-PAGEsodium dodecyl sulfate polyacrylamide gel electrophoresisSECsize exclusion chromatographySEC+IEXconcatenated SEC and IEX protein elution profilesTAIRthe Arabidopsis information resource. 1The abbreviations used are:CoIP-MScoimmunoprecipitation-mass spectrometryIEXion exchange chromatographyMappapparent mass measured by size exclusion chromatographyMcalccalculated mass of a predicted protein complex based on the proteins in clusterMmonocalculated monomer mass for a proteinP200200k × g pellet containing the microsomal fractionRappratio of the apparent mass to the calculated mass of the monomerS200soluble proteins after differential centrifugationSDS-PAGEsodium dodecyl sulfate polyacrylamide gel electrophoresisSECsize exclusion chromatographySEC+IEXconcatenated SEC and IEX protein elution profilesTAIRthe Arabidopsis information resource. or tandem affinity purification coupled with mass spectrometry (25Kim T.-H. 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Protein correlation profiling is an attractive method to analyze endogenous protein complexes as a function of their elution profiles. Protein complex composition prediction is based on the premise of "guilt by association" in which subunits of stable protein complexes coelute independent of the purification strategy. The method is enabled by the parallel protein quantification inherent to modern protein mass spectrometry and the availability of high-quality proteomes. Increased protein coverage and accurate quantification is being driven by improvements in mass spectrometry instrumentation and data analysis pipelines (13Aryal U.K. Xiong Y. McBride Z. Kihara D. Xie J. Hall M.C. Szymanski D.B. A proteomic strategy for global analysis of plant protein complexes.Plant Cell. 2014; 26: 3867-3882Crossref PubMed Scopus (41) Google Scholar, 21Aryal U.K. McBride Z. Chen D. Xie J. Szymanski D.B. Analysis of protein complexes in Arabidopsis leaves using size exclusion chromatography and label-free protein correlation profiling.J. Proteomics. 2017; 166: 8-18Crossref PubMed Scopus (37) Google Scholar, 31Kristensen A.R. Gsponer J. Foster L.J. A high-throughput approach for measuring temporal changes in the interactome.Nat. Methods. 2012; 9: 907-909Crossref PubMed Scopus (224) Google Scholar, 32McBride Z. Chen D. Reick C. Xie J. Szymanski D.B. Global analysis of membrane-associated protein oligomerization using protein correlation profiling.Mol. Cell. Proteomics. 2017; 16: 1972-1989Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar, 33Scott N.E. Rogers L.D. Prudova A. Brown N.F. Fortelny N. Overall C.M. Foster L.J. Interactome disassembly during apoptosis occurs independent of caspase cleavage.Mol. Syst. Biol. 2017; 13: 906Crossref PubMed Scopus (31) Google Scholar). In practice, protein complex composition prediction is challenging because the cell extract is a mixture of thousands to tens of thousands of monomers and complexes. Using size-based separations it is possible to measure the apparent mass of hundreds to thousands of proteins in a single experiment, and the subset that is likely to exist in an oligomeric state (13Aryal U.K. Xiong Y. McBride Z. Kihara D. Xie J. Hall M.C. Szymanski D.B. A proteomic strategy for global analysis of plant protein complexes.Plant Cell. 2014; 26: 3867-3882Crossref PubMed Scopus (41) Google Scholar, 31Kristensen A.R. Gsponer J. Foster L.J. A high-throughput approach for measuring temporal changes in the interactome.Nat. Methods. 2012; 9: 907-909Crossref PubMed Scopus (224) Google Scholar, 32McBride Z. Chen D. Reick C. Xie J. Szymanski D.B. Global analysis of membrane-associated protein oligomerization using protein correlation profiling.Mol. Cell. 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As an alternative approach, machine learning and bioinformatic algorithms that combine LC/MS profile data with gene coexpression, coevolution, and protein-protein interaction datasets have been used to make more restricted predictions about protein complex composition (36Havugimana P.C. Hart G.T. Nepusz T. Yang H. Turinsky A.L. Li Z. Wang P.I. Boutz D.R. Fong V. Phanse S. Babu M. Craig S.A. Hu P. Wan C. Vlasblom J. Dar V-u Bezginov .-NA Clark G.W. Wu G.C. Wodak S.J. Tillier E.R.M. Paccanaro A. Marcotte E.M. Emili A. A census of human soluble protein complexes.Cell. 2012; 150: 1068-1081Abstract Full Text Full Text PDF PubMed Scopus (588) Google Scholar, 37Wan C. Borgeson B. Phanse S. Tu F. Drew K. Clark G. Xiong X. Kagan O. Kwan J. Bezginov A. Chessman K. Pal S. Cromar G. Papoulas O. Ni Z. Boutz D.R. Stoilova S. Havugimana P.C. Guo X. Malty R.H. Sarov M. Greenblatt J. Babu M. Derry W.B. Tillier E.R. Wallingford J.B. Parkinson J. Marcotte E.M. Emili A. Panorama of ancient metazoan macromolecular complexes.Nature. 2015; 525: 339-344Crossref PubMed Scopus (317) Google Scholar). One way these algorithms are validated is through prediction of known protein complexes. In Arabidopsis, subunits of known, evolutionarily conserved protein complexes rarely exist as stable, fully assembled forms (32McBride Z. Chen D. Reick C. Xie J. Szymanski D.B. Global analysis of membrane-associated protein oligomerization using protein correlation profiling.Mol. Cell. Proteomics. 2017; 16: 1972-1989Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). Therefore, mass spectrometry profile data can be incongruent with orthogonal datasets that are constructed based on "golden standards" of assumed fully assembled complexes. Our goal here is to develop a protein correlation profile workflow in which imperfect but highly useful protein complex composition predictions can be made based on LC/MS profile data alone. Soluble Arabidopsis leaf extracts, enriched in soluble cytosolic and chloroplast proteins, were separated by SEC and IEX chromatography to generate thousands of elution profiles. Automated data filtering of biological replicates was used to identify and combine reproducible profiles and subject them to distance-based clustering analyses to identify groups of proteins with similar elution profiles. The intrinsic information content of the dendrogram and the behaviors of selected known proteins complexes were used to divide the dendrogram and generate specific protein complex composition predictions. An array of biochemical and genetic validation experiments demonstrates the utility of this dataset and the potential use of this method to generate systems-level knowledge about protein complex composition and dynamics. For LC-MS/MS profiling two biological replicates were used based on the high level of reproducibility between replicates. In previous studies most of the proteins had a reproducible peak between biological replicates (13Aryal U.K. Xiong Y. McBride Z. Kihara D. Xie J. Hall M.C. Szymanski D.B. A proteomic strategy for global analysis of plant protein complexes.Plant Cell. 2014; 26: 3867-3882Crossref PubMed Scopus (41) Google Scholar, 21Aryal U.K. McBride Z. Chen D. Xie J. Szymanski D.B. Analysis of protein complexes in Arabidopsis leaves using size exclusion chromatography and label-free protein correlation profiling.J. Proteomics. 2017; 166: 8-18Crossref PubMed Scopus (37) Google Scholar, 32McBride Z. Chen D. Reick C. Xie J. Szymanski D.B. Global analysis of membrane-associated protein oligomerization using protein correlation profiling.Mol. Cell. Proteomics. 2017; 16: 1972-1989Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). Ion exchange chromatography provided a high-resolution separation and 65 fractions were analyzed by mass spectrometry (analyzed on Sciex 5600 mass spectrometer). For the SEC and IEX profiling experiments that were analyzed to predict protein complex composition were analyzed on Sciex 5600 mass spectrometer. The SEC fractions that were analyzed to test for oligomerization changes in predicted AIMP1L-interactors by profiling the aimpl1 mutant were analyzed on Q Exactive mass spectrometer. For CoIP-MS pull downs three replicates were performed with antibodies against the protein of interest and negative controls and were analyzed on Q Exactive mass spectrometer. Arabidopsis thaliana ecotype Colombia was grown in tissue culture under continuous light (0.5× MS salts, 1% sucrose, 0.8% Bacto agar) for 21 days after germination (13Aryal U.K. Xiong Y. McBride Z. Kihara D. Xie J. Hall M.C. Szymanski D.B. A proteomic strategy for global analysis of plant protein complexes.Plant Cell. 2014; 26: 3867-3882Crossref PubMed Scopus (41) Google Scholar). Two grams of leaf tissue was collected and all the remaining steps were performed immediately without freezing at 4 °C on ice. The leaves were transferred to a 50 ml round bottom centrifuge tube with 7 ml of ice-cold MIB buffer (50 mm HEPES-KOH pH 7.5, 250 mm sorbitol, 50 mm KOAc, 2 mm Mg(OAc)2, 1 mm EDTA, 1 mm EGTA, 1 mm DTT, 2 mm phenyl methyl sulfonylfluoride and 1% (v/v) inhibitor mixture (160 mg/ml benzamidine-HCl, 12 mg/ml phenanthroline, 0.1 mg/ml aprotinin, 100 mg/ml leupeptin, and 0.1 mg/ml pepstatin A) for homogenization. Two 10 s bursts of a polytron (Brinkmann Instruments, Riverview, FL) homogenized the tissue. Debris was removed by filtration of the homogenate through four layers of cheesecloth. Differential centrifugation enriched the soluble proteins by spinning at 1000 × g (Beckman Avanti 30, Alanta, GA) for 10 min, 4 °C. The supernatant was enriched by pelleting membranes by ultracentrifugation at 200,000 × g for 20 min, 4 °C (Beckman Optima Ultracentrifuge). The remaining supernatant contained the crude cytosolic proteins. RuBisCO was depleted from the crude cytosolic fraction using Seppro RuBisCO spin columns according to the manufacturer's specifications (Sigma Aldrich, St. Louis, MO). Size exclusion chromatography was performed on an AKTA FPLC system (GE Life Sciences, Pittsburgh, PA) using either a Superdex increase 200 10/300 GL (GE Healthcare) or HiLoad 16/600 Superdex 200 pg column (GE Life sciences). The mobile phase was [50 mm HEPES-KOH pH 7.8, 100 mm NaCl, 10 mm MgCl2, 5% glycerol and 1 mm DTT] and flow rates were 0.6 ml/minute for the 10/300 column and 1 ml/min for the 16/600 column. Protein loading was 0.5 ml (∼1 μg total protein) for the 10/300 and 2 ml (∼4 mg total protein) for the 16/600 column. The columns were calibrated using the gel filtration kit 1000 (MWGF1000, Sigma-Aldrich) using standards ranging from 669 to 29 kDa and the void was determined using blue dextran as previously described (13Aryal U.K. Xiong Y. McBride Z. Kihara D. Xie J. Hall M.C. Szymanski D.B. A proteomic strategy for global analysis of plant protein complexes.Plant Cell. 2014; 26: 3867-3882Crossref PubMed Scopus (41) Google Scholar). Fractions were collected starting at the void to ∼5 kDa. For separation by charge using ion exchange chromatography a buffer exchange was required for effective protein binding to the solid phase. Buffer exchange was performed using Amicon ultra-15 50 ml centrifugal filters (Milipore, Burlington, MA) to exchange into 20 mm Tris/HCl pH 7.5. IEX chromatography was performed using a Dionex Ultimate 3000 UPLC (Thermo Fisher, Waltham, MA) and a PolyLC (Columbia, MD) mixed bed ion exchange column in Buffer A [20 mm Tris/HCl pH 7.5, 5% glycerol, and 0.5 mm DTT] then eluted with a 35 min linear gradient to increase the mobile phase to 50% buffer A and 50% Buffer B [20 mm Tris/HCl pH 7.5, 5% glycerol, 1.5 m NaCl and 0.5 mm DTT] and over the final 5 min the buffer composition was ramped to 25% Buffer A and 75% Buffer B. Sixty-five 500 μl fractions were collected. Proteins were separated by SDS-PAGE and visualized with Coomassie blue staining using standard procedures. Proteins were loaded by equal proportions in 1× Laemmli buffer [0.1 M Tris-HCl, pH 6.8, 1% SDS and 5% glycerol] onto 10% gels and stained with Coomassie blue [50% Methanol, 10% acetic acid and 0.0125% Coomassie blue]. For mass spectrometry analysis, proteins were digested to peptides as described in (32McBride Z. Chen D. Reick C. Xie J. Szymanski D.B. Global analysis of membrane-associated protein oligomerization using protein correlation profiling.Mol. Cell. Proteomics. 2017; 16: 1972-1989Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). Briefly, the chromatography mobile phase was removed by acetone precipitation, proteins were solubilized and denatured with urea and digested with trypsin. Peptide concentrations were measured with a BCA assay and the most concentrated sample was adjusted to have a peptide concentration of 0.2 μg/μl and an injection volume of 5 μl was analyzed by mass spectrometry. For the AB Sciex 5600, SEC and IEX samples were analyzed by LC-MS/MS as described by Aryal et al. (21Aryal U.K. McBride Z. Chen D. Xie J. Szymanski D.B. Analysis of protein complexes in Arabidopsis leaves using size exclusion chromatography and label-free protein correlation profiling.J. Proteomics. 2017; 166: 8-18Crossref PubMed Scopus (37) Google Scholar). In brief, an Eksigent nano-LC 425 HPLC (Dublin, CA) separated the peptides over a 90 min 0 to 35% acetonitrile gradient. For the AB Sciex 5600, a quadruple time-of-flight mass spectrometer operated in a data-dependent mode. For the Thermo Fisher Q Exactive high field mass spectrometer, samples were analyzed by reverse-phase HPLC-ESI-MS/MS using the Dionex UltiMate 3000 RSLC nano System coupled to the Q Exactive High Field (HF) Hybrid Quadrupole Orbitrap MS (Thermo Fisher Scientific) and a Nano- electrospray Flex ion source (Thermo Fisher Scientific). Peptides were loaded onto a trap column (300 mm x 5 mm) packed with 5 mm 100 Å PepMap C18 medium and washed using a flow rate of 5 μl/minute with 98% purified water/2% acetonitrile (ACN)/0.01% formic acid (FA) for 5 min. Peptides were separated using a reverse phase Acclaim PepMap RSLC C18 analytical column using a 120-min method at a flow rate of 300 nl/minute. The analytical column was packed with 100 Å PepMap C18 medium (Th