Pro-CrossLink. Software tool for protein cross-linking and mass spectrometry

To facilitate structural analysis of proteins and protein-protein interactions, we developed Pro-CrossUnk, a suite of software tools consisting of three programs (Figure 1), DetectShift, ldentifyXUnk, and AssignXLink. DetectShift was developed to detect ions of cross-linked peptide pairs in a mixture of O-18-labeled peptides obtained from protein proteolytic digests. The selected candidate ions of crosslinked peptide pairs subsequently undergo tandem mass spectrometric (MS/MS) analysis for sequence determination. Based on the masses of candidate ions as well as y- and b-type ions in the tandem mass spectra, IdentifyX-Link assigns the candidate ions to cross-linked peptide pairs. For an identified cross-linked peptide pair, AssignXLink generates an extensive fragment ion list, including a-, b-, c-type, x-, Y-9 Z-type, internal, and immonium ions with associated common losses of H2O, NH3, CO, and CO2, and facilitates a precise location of the cross-linked residues. Pro-CrossUnk is automated, highly configurable by the user, and applicable to many studies that map low-resolution protein structures and molecular interfaces in protein complexes.