Improved Peptide Backbone Fragmentation Is the Primary Advantage of MS-Cleavable Crosslinkers

Abstract

Proteome-wide crosslinking mass spectrometry studies have coincided with the advent of mass spectrometry (MS)-cleavable crosslinkers that can reveal the individual masses of the two crosslinked peptides. However, recently, such studies have also been published with noncleavable crosslinkers, suggesting that MS-cleavability is not essential. We therefore examined in detail the advantages and disadvantages of using the commonly used MS-cleavable crosslinker, disuccinimidyl sulfoxide (DSSO). Indeed, DSSO gave rise to signature peptide fragments with a distinct mass difference (doublet) for nearly all identified crosslinked peptides. Surprisingly, we could show that it was not these peptide masses that proved the main advantage of MS cleavability of the crosslinker, but improved peptide backbone fragmentation which reduces the ambiguity of peptide identifications. This also holds true for another commonly used MS-cleavable crosslinker, DSBU. We show furthermore that the more intricate MS3-based data acquisition approaches lack sensitivity and specificity, causing them to be outperformed by the simpler and faster stepped higher-energy collisional dissociation (HCD) method. This understanding will guide future developments and applications of proteome-wide crosslinking mass spectrometry.

Improved Analysis of Cross-Linking Mass Spectrometry Data with Kojak 2.0, Advanced by Integration into the Trans-Proteomic Pipeline

“…This creates fragment ion series that contain either the intact cross-linker and complement peptide, or simply a small mass addition equal to the remaining cross-linker after breaking at its labile bond. These predictable fragment ion products can be exploited to improve identification of cross-linked peptide sequences . When a cleavable cross-linker is specified, Kojak considers the additional product masses that occur from breakage of the labile cross-linker bonds, using these masses as additional evidence when scoring CSMs.…”

Cross-linking mass spectrometry for mapping protein complex topologies in situ

“…A common problem with the identification of cross-linked spectra matches, especially from large databases, is the unequal fragmentation of the two linked peptides, often with one fragmenting very poorly [ 60 ]. A method to address this problem is to use MS2-cleavable cross-linkers that allow much better fragmentation of both peptides [ 61 ]. Additionally, MS2-cleavable cross-linkers that cleave asymmetrically produce characteristic ‘doublet’ peaks in the MS2 spectra that can reveal the masses of the two linked peptides.…”

Cleavable crosslinkers redefined by novel MS3-trigger algorithm

“…We designed a four-step procedure that was highly likely to improve results based on our previous analysis 20 . First, isotopic envelopes in the spectrum are detected, deconvoluted to monoisotopic peaks with summed intensities, and assigned charge states ( Fig.…”