Nha-Thi Nguyen-Huynh1, Grigory Sharo2, Clément Potel1, Pélagie Fichter2, Simon Trowitzsch3, Imre Berger3, Valérie Lamour2, Patrick Schultz2, Noëlle Potier1, and Emmanuelle Leize-Wagner1
- 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
- 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
- European Molecular Biology Laboratory (EMBL), Grenoble Outstation, 6 rue Jules Horowitz, 38042 Grenoble, France
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.
CovalX Technology Used (Click each option to learn more)
The mass spectrometry data obtained during sample analysis was used to determine binding stoichiometry of a human SAGA HAT subcomplex as well as to map the protein-protein interaction network. Prior to the cross linking reaction, the complex underwent buffer exchange with 20 mM HEPES, 100 mM NaCl in 4 °C to eliminate glycerol. Bis[sulfosuccinimidyl] suberate (BS3) was used in the cross linking reaction in the normal form (d0) and isotopically labeled form (d4) in an equimolar mixture prepared by dissolving in water. The cross linking reactions were carried out on 30 μL of a 2mg/mL protein complex in a variety of cross-linker molar ratios and at two incubation times. These ratios included 1:50, 1:75 and 1:2000 with incubation times of 30 minutes or 2 hours. From these, it was determined that the optimal conditions for the reaction were 1:75 protein:crosslinker ratio for 30 minutes at room temperature. Following the reaction, it was quenched with Tris 25 mM final concentration at pH 7.5. Sinapic acid was used as the matrix (10 mg/mL in H2O/ACN/TFA (50/50/0.3)) and the spotted plate was dried at room temperature. Both the intact and cross linked complexes were analyzed using a MALDI mass spectrometer that had been modified with a CovalX HM1 detection system. From this analysis, the researchers were able to confirm the heterotetrameric stoichiometry of the specific stabilized subcomplex. A low resolution structural model of the subcomplex was also built based on the data.