Abstract
Multiple ion fragmentation methods involving collision-induced dissociation (CID), higher-energy collisional dissociation (HCD) with regular and very high energy settings, and electron-transfer dissociation with supplementary HCD (EThcD) are implemented to improve the confidence of cross-link identifications. Three different S. cerevisiae proteasome samples cross-linked by diethyl suberthioimidate (DEST) or bis(sulfosuccinimidyl)suberate (BS(3)) are analyzed. Two approaches are introduced to combine interpretations from the above four methods. Working with cleavable cross-linkers such as DEST, the first approach searches for cross-link diagnostic ions and consistency among the best interpretations derived from all four MS(2) spectra associated with each precursor ion. Better agreement leads to a more definitive identification. Compatible with both cleavable and noncleavable cross-linkers such as BS(3), the second approach multiplies scoring metrics from a number of fragmentation experiments to derive an overall best match. This significantly increases the scoring gap between the target and decoy matches. The validity of cross-links fragmented by HCD alone and identified by Kojak, MeroX, pLink, and Xi was evaluated using multiple fragmentation data. Possible ways to improve the identification credibility are discussed. Data are available via ProteomeXchange with identifier PXD018310.