Skyline documents for the article: A general method for targeted quantitative cross-linking mass spectrometry.

Varying amounts (50-1000 ng) of cross-linked BSA peptides were analyzed by LC-MS using a PRM method targeting 30 cross-linked peptide pairs. Cross-linked peptide pairs enriched from in vivo cross-linking of cultured human cells cultured in SILAC media and treated with varying concentrations of the Hsp90 inhibitor 17-AAG were analyzed by LC-MS using a PRM method to target at total of 12 precursor ions (light and heavy isotope versions for 6 cross-linked peptide pairs). Data was collected in the Bruce Laboratory at UW as well as at Cold Spring Harbor Laboratory (CSHL) during the 2016 Proteomics Course.

BSA was cross-linked with the protein interaction reporter cross-linker BDP-NHP. The protein was reduced, alkylated and digested with trypsin. The resulting peptide mixture was desalted with reversed phase solid phase extraction before LC-MS analysis. HeLa cells were cultured in SILAC media containing either isotopically light or heavy Lys and Arg. The cells were treated with varying concentrations of 17-AAG (100, 250, 500, 1000 nM) or a DMSO control for 18 h. The cells were harvested and mixed with a counterpart SILAC label DMSO control cell sample. The cells were then cross-linked with BDP-NHP followed by protein extraction with 8M urea. The protein was reduced, alkylated and digested with trypsin. Cross-linked peptides were enriched using a combination of strong cation exchange chromatography followed by avidin affinity chromatography before LC-MS analysis.