eXL-MS: An Enhanced Cross-Linking Mass Spectrometry Workflow To Study Protein Complexes
Résumé
The analysis of proteins and protein complexes by cross-linking mass spectrometry (XL-MS) has expanded in the past decade. However, mostly used approaches suffer important limitations in term of efficiency and sensitivity. We describe here a new workflow based on the advanced use of the trifunctional cross-linker NNP9. NNP9 carries an azido group allowing the quantitative and selective introduction of a biotin molecule into cross-linked proteins. The incorporation is performed by click-chemistry using an adapted version of the enhanced filter-aided sample preparation (eFASP) protocol. This protocol, based on the use of a molecular filter, allows a very high recovery of peptides after enzymatic digestion and complete removal of contaminants. This in turn offers the possibility for one to analyze very large membrane proteins solubilized in detergent. After trypsin digestion, biotinylated peptides can be easily enriched on monoavidin beads and analyzed by LC-MS/MS. The whole workflow was developed on creatine kinase in the presence of detergent. It led to a drastic improvement in the number of identified cross-linked peptides (407 vs 81), compared to the conventional approach using a gel-based separation. One great advantage of our enhanced cross-linking mass spectrometry (eXL-MS) workflow is its high efficiency, allowing the analysis of a very low amount of material (15 μg). We also demonstrate that higher-energy collision dissociation (HCD) outperforms electron-transfer/higher-energy collision dissociation (EThcD) in terms of number of cross-linked peptides identified, but EThcD leads to better sequence coverage than HCD and thus easier localization of cross-linking sites.