Accurate Retention Time Prediction Based on Monolinked Peptide Information to Confidently Identify Cross-Linked Peptides

Abstract

Cross-linking mass spectrometry methods have not been successfully applied to protein−protein interaction discovery at a proteome-wide level mainly due to the computation complexity (O (n 2 )) issue. In a previous report, we proposed a decision tree searching strategy (DTSS), which can reduce complexity by orders of magnitude. In this study, we further found that the monolinked peptides carry out the information on the retention time of the corresponding cross-linked pairs; therefore, the retention time of cross-linked peptide pairs can be predicted accurately. By utilizing the retention time as an extra filter, the false positive rate can be reduced by around 86% with a sensitivity loss of 10%. The method combined with DTSS (T-DTSS) not only benefits improving identification confidence but also leads to lower cutoff scores and facilitates substantially increasing inter-cross-link identification. T-DTSS was successfully applied to the identification of inter-cross-links obtained from Escherichia coli cell lysate cross-linked by a newly synthesized enrichable crosslinker, pDSBE. The approach can be applicable to both cleavable and noncleavable methods.

Chemical reagents for the enrichment of modified peptides in MS-based identification

“…Phenoxyl ether bond and benzyl ether bond that were supposed to be cleavable were introduced to yield the cross-linkers pDSPE (14) and pDSBE (15), respectively. 50,51 However, pDSPE ( 14) labeled peptides showed resistance to be cleaved under MS conditions. Although the cleavage of the benzyl ether bond was observed, the abundance of the signature ions of cleaved peptides was not high and only 20% of the spectra had more than one signature.…”