title: Elution profile analysis of SDS-induced subcomplexes by quantitative mass spectrometry.
abstract: Analyzing the molecular architecture of native multiprotein complexes via biochemical methods has so far been difficult and error prone. Protein complex isolation by affinity purification can define the protein repertoire of a given complex, yet, it remains difficult to gain knowledge of its substructure or modular composition. Here, we introduce SDS concentration gradient induced decomposition of protein complexes coupled to quantitative mass spectrometry and in silico elution profile distance analysis. By applying this new method to a cellular transport module, the IFT/lebercilin complex, we demonstrate its ability to determine modular composition as well as sensitively detect known and novel complex components. We show that the IFT/lebercilin complex can be separated into at least five submodules, the IFT complex A, the IFT complex B, the 14-3-3 protein complex and the CTLH complex, as well as the dynein light chain complex. Furthermore, we identify the protein TULP3 as a potential new member of the IFT complex A and showed that several proteins, classified as IFT complex B-associated, are integral parts of this complex. To further demonstrate EPASIS general applicability, we analyzed the modular substructure of two additional complexes, that of B-RAF and of 14-3-3-ε. The results show, that EPASIS provides a robust as well as sensitive strategy to dissect the substructure of large multiprotein complexes in a highly time- as well as cost-effective manner.
authors.affiliation: Division of Experimental Ophthalmology and Medical Proteome Center, Centre for Ophthalmology, University of Tübingen, 72076 Tübingen, Germany;
authors.forename: Yves
authors.initials: Y
authors.lastname: Texier
authors.forename: Grischa
authors.initials: G
authors.lastname: Toedt
authors.forename: Matteo
authors.initials: M
authors.lastname: Gorza
authors.forename: Dorus A
authors.initials: DA
authors.lastname: Mans
authors.forename: Jeroen
authors.initials: J
authors.lastname: van Reeuwijk
authors.forename: Nicola
authors.initials: N
authors.lastname: Horn
authors.forename: Jason
authors.initials: J
authors.lastname: Willer
authors.forename: Nicholas
authors.initials: N
authors.lastname: Katsanis
authors.forename: Ronald
authors.initials: R
authors.lastname: Roepman
authors.forename: Toby J
authors.initials: TJ
authors.lastname: Gibson
authors.forename: Marius
authors.initials: M
authors.lastname: Ueffing
authors.forename: Karsten
authors.initials: K
authors.lastname: Boldt
mesh_terms: D048948:14-3-3 Proteins / Q000737:chemistry / Q000302:isolation & purification; D057809:HEK293 Cells; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D013058:Mass Spectrometry / Q000191:economics / Q000379:methods*; D046912:Multiprotein Complexes / Q000737:chemistry* / Q000302:isolation & purification*; D021122:Protein Subunits / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism; D040901:Proteomics; D048493:Proto-Oncogene Proteins B-raf / Q000737:chemistry / Q000302:isolation & purification; D012967:Sodium Dodecyl Sulfate
chemical_list: D048948:14-3-3 Proteins; D047908:Intracellular Signaling Peptides and Proteins; D046912:Multiprotein Complexes; D021122:Protein Subunits; D011506:Proteins; C116465:TULP3 protein, human; D012967:Sodium Dodecyl Sulfate; D048493:Proto-Oncogene Proteins B-raf