Fig. 4: Unique properties of DOPA2 compared with DSS.

a SDS-PAGE of DOPA2 or DSS-cross-linked BSA under different reaction times at 0 °C. b SDS-PAGE of DOPA2 or DSS-cross-linked BSA under different pH conditions. One cross-linking experiment was performed for each protein sample in (a, b). c The log₂ transformed MS1 peak intensity ratios of cross-linked products at pH 3.0 vs. pH 7.4. The synthesized peptides TR-8, VR-7, KR-7, and GR-11 were further used to evaluate the reactivity of DOPA2 at low pH without the influence of tertiary structure. The N-terminus of GR-11 was blocked by dimethylation, and thus only the lysine residue could be cross-linked. “Intact peptide” refers to free peptides without cross-linking. “X-link” refers to the situation wherein two peptides are linked with one molecule of DOPA2. “Mono-link” refers to a peptide that has been modified but is not cross-linked by a cross-linker. n = 4 biologically independent samples. d, e SDS-PAGE of DOPA2 or DSS-cross-linked BSA in the presence of the indicated denaturants (urea or GdnHCl). The experiments were repeated twice independently with similar results. f The log₂ transformed MS1 peak intensity ratios of cross-linked products at GdnHCl (6 M) vs. a physiological buffer (HEPES, pH 7.4). Similar to (c), the synthesized peptides VR-7 and GR-11 were used to further evaluate the reactivity of DOPA2 under high concentrations of GdnHCl without the influence of tertiary structure. n = 2 biologically independent samples. The label of products is the same as in (c). Source data for a-f are provided as a Source Data file.