Cross-linking mass spectrometry (XL-MS) has evolved into a pivotal technique for probing protein interactions, with recent ad-vancements significantly enhancing its analytical potential. This technical note presents advancements in cross-linking mass spec-trometry (XL-MS) through the implementation of Parallel Accumulation-Serial Fragmentation (PASEF) on timsTOF instruments, enhancing the detection and analysis of protein interactions. Addressing the challenges in XL-MS, such as the interpretation of complex spectra, low abundance of cross-linked peptides (XL-peptides), and data acquisition biases, the 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 method is described for processing DDA-PASEF data using Bruker software, enabling the conversion of this data into a format compatible with MeroX and Skyline tools. This approach, demonstrated with a bovine serum albumin (BSA) case study, significantly improves the identification and understanding of protein interactions, equipping the cross-linking community to overcome current analytical limitations.

This study showcases the development of a XL-MS data analysis workflow with MeroX and Skyline. Bovine serum albumin (BSA) was cross-linked with disuccinimidyl dibutyric urea (DSBU) and digested with Trypsin using a suspension trapping (S-Trap) digestion protocol. The cross-linked BSA-DSBU tryptic digest was analyzed by LC-TIMS-MS/MS with a timsTOF Pro using parrallel accumulation-serial fragmentation (PASEF) in DDA mode. Raw LC-TIMS-MS/MS data was pre-processed with DataAnalysis to create peak lists in MGF format. The MGF files were used as input for cross-linked peptides identification with MeroX 2.0. MeroX results were converted into proXL format. The proXL files and MGF files were used as input for creating spectral libraries with Skyline. These spectral libraries allowed a peptide-centric interrogation of XL-MS data of DDA and DIA data.

A 10 μM bovine serum albumin (BSA) solution in 50 mM 2-[4-(2-hydroxyethyl)-piperazine-1-yl]ethanesulfonic acid (HEPES) (pH 7.5) underwent cross-linking at room temperature for 1 hour using a 50-fold molar excess of DSBU, freshly dissolved in DMSO at 25 mM. Alongside, a negative control without DSBU was prepared. Post cross-linking, three 80 μg aliquots from each sample were dried using a SpeedVac concentrator. Sample preparation, including protein digestion, was performed using Suspension-Trapping (S-Trap™; Protifi) as per the manufacturer’s guidelines.