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Cross-linking mass spectrometry is an emerging structural biology technique. Almost exclusively, the analyzer of choice for such an experiment has been the Orbitrap. We present an optimized protocol for the use of a Synapt G2-Si for the analysis of cross-linked peptides. We first tested six different energy ramps and analyzed the fragmentation behavior of cross-linked peptides identified by xQuest. By combining the most successful energy ramps, cross-link yield can be increased by up to 40%. When compared to previously published Orbitrap data, the Synapt G2-Si also offers improved fragmentation of the β peptide. In order to improve cross-link quality control we have also developed ValidateXL, a programmatic solution that works with existing cross-linking software to improve cross-link quality control.

Optimization Workflow for the Analysis of Cross-Linked Peptides Using a Quadrupole Time-of-Flight Mass Spectrometer