New insights on the disulfide bond status in soy proteins from mass spectrometry-pLink2 analysis and molecular docking

1. Native soy protein contains four SS-linked fractions: three polymers and AB band. 2. Quantitative analysis revealed more SS in polymers, with minor changes in AB band. 3. SS connectivities were analyzed by pLink2 and showed more heterogeneous cross-links. 4. Key reactive sites for newly formed SSs were detected among four SS-linked fractions. 5. The feasibility of new SSs was confirmed by AutoDock Vina and AlphaFold-Multimer. Disulfide bonds (SSs) are critical for stabilizing protein structures and maintaining functional properties. To obtain a better understanding of SS patterns in native soy proteins, pLink2-assisted LC-MS/MS was conducted to analyze the SS quantities and connectivities. There were 4 major disulfide-linked protein fractions—polymer-1, polymer-2, polymer-3, and AB band in soy proteins. Quantitative analysis revealed that the determined SS contents in polymers were markedly higher than those of theoretical disulfide bonds (SSs*), with a minor change in the AB band. Compared to the reported X-ray crystallography observation, much more heterogeneous SS connectivities were found. Specifically, key reactive sites for new SS formation included Cys1 st , Cys7 th , and Cys8 th in Group I and Cys4 th and Cys5 th in Group II, with the SS Cys1 st -Cys7 th displaying the highest abundance. Finally, molecular docking confirmed the feasibility of the rearranged SSs. This study disclosed formerly unknown complexity and heterogeneity, thereby deepening molecular-level understanding of soy proteins.