Bruce - PRM_XL_manuscript

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A general method for targeted quantitative cross-linking mass spectrometry

  • Organism: Bovine, Human
  • Instrument: Q-Exactive Plus, Q-Exactive Plus HF
  • SpikeIn: No
Chemical cross-linking mass spectrometry (XL-MS) provides protein structural information by identifying covalently linked proximal amino acid residues on protein surfaces. The information gained by this technique is complementary to other structural biology methods such as x-ray crystallography, NMR and cryo-electron microscopy. The extension of traditional quantitative proteomics methods with chemical cross-linking can provide information on the structural dynamics of protein structures and protein complexes. The identification and quantitation of cross-linked peptides remains challenging for the general community, requiring specialized expertise ultimately limiting more widespread adoption of the technique. We describe a general method for targeted quantitative mass spectrometric analysis of cross-linked peptide pairs utilizing PRM and Skyline for analysis. We demonstrate the utility and robustness of the method with a cross-laboratory study analyzing cross-linked bovine serum albumin (BSA) samples and cross-linked SILAC labeled cultured cells treated with varying concentrations of the Hsp90 inhibitor 17-AAG. This advance provides an easy to use resource so that any lab with access to a LC-MS system capable of performing targeted quantitative analysis can quickly and accurately measure dynamic changes in protein structure and protein interactions.
Experiment Description
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.
Sample Description
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.
Created on 9/21/16, 3:45 PM