biostudies-literatureRojas Echeverri JCMartin-Luther-Universit?t Halle-WittenbergDeutsche ForschungsgemeinschaftUniversity of WashingtonBundesministerium f?r Wirtschaft und EnergieNational Institute of General Medical SciencesRegion of Saxony-AnhaltNIGMS NIH HHS7373-7379https://www.ebi.ac.uk/biostudies/studies/S-EPMC11099889biostudies-literatureUnknown96(19)Cross-linking mass spectrometry (XL-MS) has evolved into a pivotal technique for probing protein interactions. This study describes the implementation of Parallel Accumulation-Serial Fragmentation (PASEF) on timsTOF instruments, enhancing the detection and analysis of protein interactions by XL-MS. Addressing the challenges in XL-MS, such as the interpretation of complex spectra, low abundant cross-linked peptides, and a data acquisition bias, our current study integrates a peptide-centric approach for the analysis of XL-MS data and presents the foundation for integrating data-independent acquisition (DIA) in XL-MS with a vendor-neutral and open-source platform. A novel workflow is described for processing data-dependent acquisition (DDA) of PASEF-derived information. For this, software byAnalytical chemistryA Workflow for Improved Analysis of Cross-Linking Mass Spectrometry Data Integrating Parallel Accumulation-Serial Fragmentation with MeroX and Skyline.PMC11099889INST 271/404-1 FUGGRTG 2467 - 391498659UWPR95794R24 GM141156CRC 1423 - 421152132KK5096401SK0INST 271/405-1 FUGGTanzler DShulman NRiffle MMacLean BXHause FIhling CHIacobucci CSinz ARojas Echeverri JCfalseA Workflow for Improved Analysis of Cross-Linking Mass Spectrometry Data Integrating Parallel Accumulation-Serial Fragmentation with MeroX and Skyline.Cross-linking mass spectrometry (XL-MS) has evolved into a pivotal technique for probing protein interactions. This study describes the implementation of Parallel Accumulation-Serial Fragmentation (PASEF) on timsTOF instruments, enhancing the detection and analysis of protein interactions by XL-MS. Addressing the challenges in XL-MS, such as the interpretation of complex spectra, low abundant cross-linked peptides, and a data acquisition bias, our current study integrates a peptide-centric approach for the analysis of XL-MS data and presents the foundation for integrating data-independent acquisition (DIA) in XL-MS with a vendor-neutral and open-source platform. A novel workflow is described for processing data-dependent acquisition (DDA) of PASEF-derived information. For this, software by2024-01-01T00:00:00Z2024 May2026-04-08T19:50:50.619Z2026-04-08T14:33:42.723ZS-EPMC110998893869681910.1021/acs.analchem.4c00829