Use of Multiple Ion Fragmentation Methods to Identify Protein Cross-Links and Facilitate Comparison of Data Interpretation Algorithms

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.