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Protein Sci. 2015 Aug;24(8):1232-46. doi: 10.1002/pro.2676. Epub 2015 Apr 14.

Chemical cross-linking and mass spectrometry to determine the subunit interaction network in a recombinant human SAGA HAT subcomplex.

Author information

1
Laboratoire de Spectrométrie de Masse des Interactions et des Systèmes (LSMIS) UMR 7140 CNRS/Université de Strasbourg - "Chimie de la Matière Complexe", 1 Rue Blaise Pascal, 67008, Strasbourg, France.
2
Integrated Structural Biology Department, Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), UMR 7104, INSERM U964, 1 rue Laurent Fries, 67404, Illkirch, France.
3
European Molecular Biology Laboratory (EMBL), Grenoble Outstation, 6 rue Jules Horowitz, 38042 Grenoble, France.

Abstract

Understanding the way how proteins interact with each other to form transient or stable protein complexes is a key aspect in structural biology. In this study, we combined chemical cross-linking with mass spectrometry to determine the binding stoichiometry and map the protein-protein interaction network of a human SAGA HAT subcomplex. MALDI-MS equipped with high mass detection was used to follow the cross-linking reaction using bis[sulfosuccinimidyl] suberate (BS3) and confirm the heterotetrameric stoichiometry of the specific stabilized subcomplex. Cross-linking with isotopically labeled BS3 d0-d4 followed by trypsin digestion allowed the identification of intra- and intercross-linked peptides using two dedicated search engines: pLink and xQuest. The identified interlinked peptides suggest a strong network of interaction between GCN5, ADA2B and ADA3 subunits; SGF29 is interacting with GCN5 and ADA3 but not with ADA2B. These restraint data were combined to molecular modeling and a low-resolution interacting model for the human SAGA HAT subcomplex could be proposed, illustrating the potential of an integrative strategy using cross-linking and mass spectrometry for addressing the structural architecture of multiprotein complexes.

KEYWORDS:

SAGA HAT subcomplex; cross-linking mass spectrometry; protein interaction; protein-; proteomics

PMID:
25753033
PMCID:
PMC4534174
DOI:
10.1002/pro.2676
[Indexed for MEDLINE]
Free PMC Article

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