Pro-CrossLink. Software Tool for Protein Cross-Linking and Mass Spectrometry

Abstract

To facilitate structural analysis of proteins and protein-protein interactions, we developed Pro-CrossLink, a suite of software tools consisting of three programs (Figure 1), DetectShift, IdentifyXLink, and AssignXLink. DetectShift was developed to detect ions of cross-linked peptide pairs in a mixture of 18O-labeled peptides obtained from protein proteolytic digests. The selected candidate ions of cross-linked 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, IdentifyXLink 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-, 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-CrossLink is automated, highly configurable by the user, and applicable to many studies that map low-resolution protein structures and molecular interfaces in protein complexes.

Identification of cross-linked peptides from large sequence databases

“…Second, the computational challenge is foremost the combinatorial explosion of the search space that results when all peptide:peptide combinations in a database are considered. Consequently, currently available cross-link analysis programs 1,3,[10][11][12] are limited to a sequence database size of only a few proteins.…”

Structural Analysis of Multiprotein Complexes by Cross-linking, Mass Spectrometry, and Database Searching

“…Standard database search tools cannot create a list of candidate cross-linked peptides based on the observed mass. A number of dedicated programs consider pairs of peptides contributing together to the observed mass (7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17). The candidates then need to be validated on the basis of their match to fragmentation spectra.…”

“…The candidates then need to be validated on the basis of their match to fragmentation spectra. Currently this requires screening the spectra either completely manually or through software assistance (13,15,16,18). No scoring system or algorithm has yet been developed to replace human intervention.…”

Impact of proline and aspartic acid residues on the dissociation of intermolecularly crosslinked peptides

“…Once distinguished and identified, collisionally activated dissociation (CAD) experiments on these species are performed to sequence the peptide or peptides. Structural assignment of the crosslinked peptides is still not routine despite the emergence of several sequencing algorithms [17][18][19][20][21][22]. In fact, one of the main pitfalls of mass spectrometric strategies for examination of crosslinked peptides is the lack of comprehensive rules for interpretation of the product ion spectra.…”