Disuccinimidyl sulfoxide

For NHS-ester-based cross-linking (XL) reactions, all aliquots of XL reagents were freshly diluted in anhydrous DMSO (Invitrogen™ by Thermo Fisher Scientific, Rockford, IL, USA). Following reagents were used: PhoX (Disuccinimidyl Phenyl Phosphonic Acid, Bruker); DSAU (Disuccinimidyl diacetic urea, CF Plus Chemicals, Brno-Řečkovice, Czech Republic); DSSO (Disuccinimidyl sulfoxide, Thermo Fisher Scientific, Rockford, IL, USA); DSBU (Disuccinimidyl dibutyric urea, CF Plus Chemicals, Brno-Řečkovice, Czech Republic). BSA, ADH, GLDH and 20S samples were each split in six aliquots and incubated with 25, 100, or 400 molar excess of each reagent. All samples were additionally cross-linked with 800 and 100 molar excesses of DSBU. For R2SP cross-linking, stock solution was split into 20 aliquots subsequently reacted with PhoX, DSAU, DSSO, DSBU at molar excesses of 25, 50, 100, 200, 400.
For formaldehyde cross-linking, GLDH and 20 S proteasome at 1 mg/mL were incubated by adding a formaldehyde 37% stock solution (Merck KGaA, Darmstadt, Germany) diluted to 0.05%, 0.1%, 0.2%, 0.5% and 1% (vol/vol final) for 20 min at room temperature^{41 (link)}.
All XL reactions were carried for all samples at room temperature (20 °C) for 45 min, and quenched with Tris HCl (15 mM final concentration) for 20 min. An aliquot of each non-XL control and XL sample was kept for SDS-PAGE migration.