Mass spectrometry analysis of DSBU cross‐linked protein complexes

Cross‐linked proteins were digested to generate peptides for MS analysis. Initially, proteins were reduced and alkylated in a buffer containing 5% SDS, 10 mM TCEP, and 11 mM CAA in 100 mM Tris–HCl pH 8.5 for 10 min at 95°C and precipitated on MagResyn HILIC magnetic particles (Resyn Biosciences) in 70% acetonitrile for 20 min and washed with 95% acetonitrile and 70% ethanol before on‐bead digestion with Lys‐C and trypsin in 50 mM ammonium bicarbonate pH 8.0 overnight (Batth et al, 2019). The resulting peptides were desalted on Sep‐Pak tC18 cartridges (Waters Corporation) and subjected to direct MS‐analysis (10% of the sample) or further enriched for multiple charged cross‐linked peptides on mixed‐mode C18/SCX cartridges (Oasis MCX, Waters Corporation) generating four fractions for MS analysis as previously described (Iacobucci et al, 2018). Cross‐linked peptides were analyzed by an Easy nanoLC system coupled to an Orbitrap Exploris 480 mass spectrometer (Thermo Scientific) using data‐dependent acquisition of ions with a charge state between 3 and 8 and HCD fragmentation using stepped normalized collision energy (NCE) of 27, 30 and 33. Interpeptide cross‐links were identified based on their signature doublet signals from the cleavable DSBU cross‐linker as implemented in the RISEUP mode of the Program MeroX version 2.0 (Götze et al, 2019). The identified cross‐links were evaluated by scores for peptide pair identifications and cross‐link position assignments within the peptide sequences.