Prosit-XL: enhanced cross-linked peptide identification by fragment intensity prediction to study protein interactions and structures.

It has been shown that integrating peptide property predictions such as fragment intensity into the scoring process of peptide spectrum match can greatly increase the number of confidently identified peptides compared to using traditional scoring methods. Here, we introduce Prosit-XL, a robust and accurate fragment intensity predictor covering the cleavable (DSSO/DSBU) and non-cleavable cross-linkers (DSS/BS3), achieving high accuracy on various holdout sets with consistent performance on external datasets without fine-tuning. Due to the complex nature of false positives in XL-MS, an approach to data-driven rescoring was developed that benefits from Prosit-XL's predictions while limiting the overestimation of the false discovery rate (FDR). After validating this approach using two ground truth datasets consisting of synthetic peptides and proteins, we applied Prosit-XL on a proteome-scale dataset, demonstrating an up to ~3.4-fold improvement in PPI discovery compared to classic approaches. Finally, Prosit-XL was used to increase the coverage and depth of a spatially resolved interactome map of intact human cytomegalovirus virions, leading to the discovery of previously unobserved interactions between human and cytomegalovirus proteins.