title: Multivariate proteomic profiling identifies novel accessory proteins of coated vesicles.
abstract: Despite recent advances in mass spectrometry, proteomic characterization of transport vesicles remains challenging. Here, we describe a multivariate proteomics approach to analyzing clathrin-coated vesicles (CCVs) from HeLa cells. siRNA knockdown of coat components and different fractionation protocols were used to obtain modified coated vesicle-enriched fractions, which were compared by stable isotope labeling of amino acids in cell culture (SILAC)-based quantitative mass spectrometry. 10 datasets were combined through principal component analysis into a "profiling" cluster analysis. Overall, 136 CCV-associated proteins were predicted, including 36 new proteins. The method identified >93% of established CCV coat proteins and assigned >91% correctly to intracellular or endocytic CCVs. Furthermore, the profiling analysis extends to less well characterized types of coated vesicles, and we identify and characterize the first AP-4 accessory protein, which we have named tepsin. Finally, our data explain how sequestration of TACC3 in cytosolic clathrin cages causes the severe mitotic defects observed in auxilin-depleted cells. The profiling approach can be adapted to address related cell and systems biological questions.
authors.affiliation: Cambridge Institute for Medical Research, Wellcome Trust/Medical Research Council Building, University of Cambridge, Cambridge CB2 0XY, England, UK. ghhb2@cam.ac.uk
authors.forename: Georg H H
authors.initials: GH
authors.lastname: Borner
authors.forename: Robin
authors.initials: R
authors.lastname: Antrobus
authors.forename: Jennifer
authors.initials: J
authors.lastname: Hirst
authors.forename: Gary S
authors.initials: GS
authors.lastname: Bhumbra
authors.forename: Patrycja
authors.initials: P
authors.lastname: Kozik
authors.forename: Lauren P
authors.initials: LP
authors.lastname: Jackson
authors.forename: Daniela A
authors.initials: DA
authors.lastname: Sahlender
authors.forename: Margaret S
authors.initials: MS
authors.lastname: Robinson
mesh_terms: D033942:Adaptor Proteins, Vesicular Transport; D002455:Cell Division; D002478:Cells, Cultured; D002853:Chromatography, Liquid; D022163:Clathrin-Coated Vesicles / Q000737:chemistry* / Q000378:metabolism; D019295:Computational Biology; D006367:HeLa Cells; D006801:Humans; D013058:Mass Spectrometry; D040901:Proteomics; D053719:Tandem Mass Spectrometry
chemical_list: D033942:Adaptor Proteins, Vesicular Transport; C000607390:tepsin protein, human