Abstract
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.
Highlights
Cross-linking mass spectrometry (XLMS) can be used to gain structural insights into proteins and complexes that cannot be studied by high-resolution structural techniques.[1,2] Cross-linking is based on the covalent bonding of two amino acids that lie within a certain proximity
The resulting approach allows quadrupole time-of-flight analysers (QTOFs) data to be analyzed with existing cross-linking software, xQuest,[13] with minimal adaptations
In contrast to data collected from Orbitrap analyzers[14] we demonstrate when using the slow de-isotoping algorithm during data processing at both the precursor and fragment ion level
Summary
Cross-linking mass spectrometry (XLMS) can be used to gain structural insights into proteins and complexes that cannot be studied by high-resolution structural techniques.[1,2] Cross-linking is based on the covalent bonding of two amino acids that lie within a certain proximity. In contrast to data collected from Orbitrap analyzers[14] we demonstrate when using the slow de-isotoping algorithm during data processing at both the precursor and fragment ion level The mid energy ramp is the most reproducible and identifies the most high-scoring cross-links: 103, 111, and 131 in the respective triplicate analysis.
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