Comparison of noncleavable crosslinker BS3 to the MS-cleavable
crosslinker DSSO. (a) Example MS2 spectrum of a high-scoring CSM identified
in both datasets. (Top) CSM from the BS3 dataset. (Bottom) The same
peptide m/z-species identified in
the DSSO dataset. Unique fragments are highlighted in bold. (b) Sequence
coverage of all, linear and link site-containing fragments of all
common CSMs (n = 12919). For DSSO, link site-containing
fragments are additionally separated into fragments containing the
full second peptide (+P) or only the cleaved crosslinker stub (A/S/T).
Boxplots depict the median (middle line), upper and lower quartiles
(boxes), and 1.5 times the interquartile range (whiskers). Asterisks
indicate significance calculated by a two-sided Wilcoxon signed rank
(p-value < 0.0001: ****). (c) Target–target
and target–decoy score distributions of heteromeric CSMs for
BS3 and DSSO. Scores were normalized to their respective score cutoff
at 10% FDR. (d) Number of heteromeric CSMs passing 5% CSM-level FDR
for BS3 and DSSO. As a control, DSSO was additionally searched as
a noncleavable crosslinker and also filtered for the presence of peptide
doublets.