Elution profile analysis of SDS-induced subcomplexes by quantitative mass spectrometry

Yves Texier*, Grischa Toedt, Matteo Gorza, Dorus A. Mans, Jeroen Van Reeuwijk, Nicola Horn, Jason Willer, Elias Nicholas Katsanis, Ronald Roepman, Toby J. Gibson, Marius Ueffing, Karsten Boldt

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

23 Scopus citations

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-. The results show, that EPASIS provides a robust as well as sensitive strategy to dissect the substructure of large multiprotein complexes in a highly timeas well as cost-effective manner.

Original languageEnglish (US)
Pages (from-to)1382-1391
Number of pages10
JournalMolecular and Cellular Proteomics
Volume13
Issue number5
DOIs
StatePublished - May 2014

ASJC Scopus subject areas

  • Analytical Chemistry
  • Biochemistry
  • Molecular Biology

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