Figure 3.

Isotope pattern of crosslinked peptides allows distinction between true-positive and false-positive crosslinks. A: The isotope-labeled crosslinking reagent DTSSP (Creative Molecules). B: Example of an isotope pattern with a 1:0:1 ratio for the +0, +4, and +8 Da triplet. The crosslink reflects a crosslinking event in the native protein. C: Example of an isotope pattern with a 1:2:1 ratio for the +0, +4, and +8 Da triplet. A crosslink that has been exclusively formed due to disulfide bond scrambling will have the 1:2:1 signature as a hallmark, and should be discarded as a false positive. D: Example of an isotope pattern with a 1:X:1 ratio, where in this case X = 0.38. This signal is predominantly originating from a crosslink that reflects a crosslinking event between the lysine residues in the peptides DK₁₅₅IK and TK₁₇₃VER that are in close proximity in the native protein, but is overlaid with a false-positive crosslink that has formed by scrambling. All peaks displaying an isotope pattern with less of the +4 Da peak than 1:2:1 reflect true crosslinks. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]