Identification of Cross-Linked Peptides by High-Resolution Precursor Ion Scan

摘要

Chemical cross-linking coupled to mass spectrometrynanalysis has become a realistic alternative to the study ofnproteins structure and interactions, especially when thesensystems are not amenable to high-resolution techniquesnsuch as protein crystallography or nuclear magneticnresonance. One of the main bottlenecks of this approachnrelies on the detection of cross-linked peptides, as theynare usually present in substoichiometric amounts inncomplex samples. It was shown that one of the mainnfragmentation pathways of disuccinimidyl suberate (DSS)ncross-linked peptides yields diagnostic ions, whose structurenis composed of a rearranged lysine side chain andnthe spacer arm of the linker. In this report, we demonstratenthe feasibility of detecting these modified peptidesnbased on a precursor ion scan in a quadrupole time-offlightn(Q-TOF) instrument. It was shown that the fragmentationnof nonmodified tryptic peptides hardly generatesnions with the same nominal mass of the diagnostic ions,nmaking the precursor ion scan very specific to N-hydroxysuccinimiden(NHS)-based cross-linkers. Moreover, thenexperimental setup is the same as in the case of a regularncross-linking experiment, not demanding any additionalnexperimental steps that would increase sample handling.nThe results obtained with protein samples allowed us tonpropose an algorithm that could be implemented in ansoftware to process data from cross-linking experimentsnin an automated and high-throughput way.