Fig. 1. Considerations for crosslinked PPI-FDR and experimental workflow.

a For matches within the same protein sequence (with a non-directional crosslinker¹⁷), a crosslink from A1 to A2 is indistinguishable from A2 to A1 (theoretically possible search space shown as purple triangles). In contrast, heteromeric matches are not symmetrical, and therefore occupy a larger random space (green squares). b Fraction of decoys in random picks of 100 self and 100 heteromeric CSMs from the search output before any FDR filtering (random picks, n = 20, i.e. ten per crosslinker dataset). Error bars show standard deviation from the mean. Source data are provided as a Source Data file. c Schematic showing error increase when merging crosslinked residue pairs to PPIs. Proteins are indicated as circles; blue and red lines represent true and false linkages, respectively. d Experimental workflow. E. coli lysate was separated and crosslinked in individual high molecular weight fractions, pooled again to simulate a complex mixture and analyzed by mass spectrometry. Quantitative proteomics of uncrosslinked fractions provided protein coelution data.