Fig. 4: Benchmarking of XlinkX PD, xiSEARCH/xiFDR and software processing times.
a–c, Interprotein CSMs, ResPairs and PPI identifications when comparing Scout and XlinkX PD. True-positive identifications by XlinkX PD are shown in light brown (540-protein database) and dark brown (4,000-protein database). The Scout numbers (blue diamonds) are the same as in Fig. 3. In addition to using our standard search parameters, XlinkX PD identification were postprocessed using a static score cutoff (‘default’) (a), score cutoffs derived from the highest scoring CSM-level decoy in every analysis (‘dynamic’) (b) and score cutoffs set to filter XlinkX PD results to 1% empirical FDR (c). The XlinkX score cutoffs are displayed below the bars. Both Scout and XlinkX PD considered K as the only cross-linking site. d, Interprotein CSMs, ResPairs and PPI identifications when comparing Scout and xiSEARCH. For Scout, results were filtered at 1% software-defined FDR on all levels. For xiSEARCH/xiFDR, following the developer’s recommendation, a 1% software-defined FDR was applied only on the PPI level using boost between proteins (xiFDR) and reported are the resulting PPIs together with their corresponding CSMs and ResPairs. Scout and xiSEARCH were run using their default parameters, respectively, with KSTY as the possible reaction sites for the cross-linking reagent. In a–d, the framed percentage numbers indicate the final empirical FDR and yellow bars show false-positive identifications, violating the mixing scheme of our XL-MS standard. e, Processing time in minutes (min) using different search engines on the benchmarking dataset with a 1% software-defined FDR cutoff on a computer with 512 GB RAM and powered by dual Intel(R) Xeon(R) Gold 6136 CPUs operating at 3.00 GHz. xiSEARCH/xiFDR did not run to completion on this hardware setup when using the full benchmarking dataset. Therefore, a separate Scout versus xi speed comparison using only four RAW files was performed and is shown in Extended Data Fig. 3b.
