Data Analysis Workflow

1) Raw Bruker DDA-PASEF files are processed with DataAnalysis to obtain summed and filtered fragment ion spectra written in peak lists with a Mascot generic format. 2) The peak lists are then used for identification of cross-linked peptides with MeroX 2.0. 3) The native MeroX results are converted into a standard protein cross-linking (ProXL) XML database scheme. 4) The peak list files and the ProXL XML files are used to create a data dependent acquisition (DDA) spectral library. This spectral library is used to guide Skyline for the creation of extracted ion chromatograms (EICs) of the proposed cross-linked peptides. 5) The Skyline document is refined accounting for other analyte-specific properties (e.g.., isotopic pattern, peak shape, isotope co-elution, consistent chromatographic retention, etc.) accessible with Skyline. At this point data analysis can be stopped and report results or, alternatively, the refined documents can then be used to optimize DDA-PASEF experiments, design and export PRM-PASEF experiments, or serve as a template for analysis of DIA-PASEF data using DDA spectral libraries.