title: Discovery of cell compartment specific protein-protein interactions using affinity purification combined with tandem mass spectrometry.
abstract: Affinity purification combined with tandem mass spectrometry (AP-MS/MS) is a well-established method used to discover interaction partners for a given protein of interest. Because most AP-MS/MS approaches are performed using the soluble fraction of whole cell extracts (WCE), information about the cellular compartments where the interactions occur is lost. More importantly, classical AP-MS/MS often fails to identify interactions that take place in the nonsoluble fraction of the cell, for example, on the chromatin or membranes; consequently, protein complexes that are less soluble are underrepresented. In this paper, we introduce a method called multiple cell compartment AP-MS/MS (MCC-AP-MS/MS), which identifies the interactions of a protein independently in three fractions of the cell: the cytoplasm, the nucleoplasm, and the chromatin. We show that this fractionation improves the sensitivity of the method when compared to the classical affinity purification procedure using soluble WCE while keeping a very high specificity. Using three proteins known to localize in various cell compartments as baits, the CDK9 subunit of transcription elongation factor P-TEFb, the RNA polymerase II (RNAP II)-associated protein 4 (RPAP4), and the largest subunit of RNAP II, POLR2A, we show that MCC-AP-MS/MS reproducibly yields fraction-specific interactions. Finally, we demonstrate that this improvement in sensitivity leads to the discovery of novel interactions of RNAP II carboxyl-terminal domain (CTD) interacting domain (CID) proteins with POLR2A.
authors.affiliation: McGill Centre for Bioinformatics and School of Computer Science, McGill University, Montréal, Québec H3A 2B4, Canada.
authors.forename: Mathieu
authors.initials: M
authors.lastname: Lavallée-Adam
authors.forename: Justine
authors.initials: J
authors.lastname: Rousseau
authors.forename: Céline
authors.initials: C
authors.lastname: Domecq
authors.forename: Annie
authors.initials: A
authors.lastname: Bouchard
authors.forename: Diane
authors.initials: D
authors.lastname: Forget
authors.forename: Denis
authors.initials: D
authors.lastname: Faubert
authors.forename: Mathieu
authors.initials: M
authors.lastname: Blanchette
authors.forename: Benoit
authors.initials: B
authors.lastname: Coulombe
mesh_terms: D002451:Cell Compartmentation / Q000235:genetics / Q000502:physiology; D002467:Cell Nucleus / Q000378:metabolism; D002846:Chromatography, Affinity / Q000379:methods*; D003593:Cytoplasm / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D042925:Positive Transcriptional Elongation Factor B / Q000378:metabolism; D011485:Protein Binding; D011506:Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012319:RNA Polymerase II / Q000737:chemistry / Q000378:metabolism; D053719:Tandem Mass Spectrometry / Q000379:methods*; D014158:Transcription, Genetic
chemical_list: D011506:Proteins; D042925:Positive Transcriptional Elongation Factor B; D012319:RNA Polymerase II; D012321:DNA-Directed RNA Polymerases