A Workflow for Improved Analysis of Cross-Linking Mass Spectrometry Data Integrating Parallel Accumulation-Serial Fragmentation with MeroX and Skyline

Abstract

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 by Bruker Daltonics is used, enabling the conversion of these data into a format that is compatible with MeroX and Skyline software tools. Our approach significantly improves the identification of cross-linked products from complex mixtures, allowing the XL-MS community to overcome current analytical limitations.

Figure 1

Data analysis workflow. Raw LC-TIMS-MS/MS data was preprocessed with DataAnalysis to create peak lists in MGF format. The MGF files were used as input for XL 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.

Figure 2

Evaluation of identification reproducibility of precursor ions associated with. UpSet plot representing the overlap of identified precursor ions (top panel) across replicate injections of a pooled digest of cross-linked BSA with DSBU. The identified precursors per replicate are contrasted with a list validated using results from all measurements in this data set. Blue highlight corresponds to the precursor ions carrying XSMs only in “Low CE 4” sample. Extracted ion chromatograms of the [M+3H]³⁺ ion of the KQTALVELLK-KFWGK-(DSBU@1,1) XL (middle panel) which was identified by MeroX only in replicate “Low CE 4” and corresponding XSM (bottom panel).