We developed a high-throughput mass spectrometry method, pLink-SS (http://pfind.ict.ac.cn/software/pLink/2014/pLink-SS.html), for precise identification of disulfide-linked peptides. Using pLink-SS, we mapped all native disulfide bonds of a monoclonal antibody and ten standard proteins. We performed disulfide proteome analyses and identified 199 disulfide bonds in Escherichia coli and 568 in proteins secreted by human endothelial cells. We discovered many regulatory disulfide bonds involving catalytic or metal-binding cysteine residues.
We thank the National BioResource Project of Japan for providing bacterial strains; the antibody center of National Institute of Biological Sciences, Beijing, for the purified anti–DAF-16 IgG2 antibody; and X.-Z. Dong, A. Hühmer, D. Horn and Z. Hao for bringing the disulfide-bond problem to our attention and for encouragement. We also thank all members of the pFind group, X. Xiong, Y. Xia, S. Chen, K. Ye, Y. Zhou and members of the Dong lab for discussion and experimental support. This work was funded by the National Scientific Instrumentation Grant Program (2011YQ09000506 to M.-Q.D.), National Natural Science Foundation of China (grant no. 21475141 to S.-M.H.), Ministry of Science and Technology of China (973 grants 2013CB911203, 2012CB910602 to R.-X.S. and 2010CB912701 to S.-M.H.), CAS Knowledge Innovation Program (grant #KGCX1-YW-13 and ICT-20126033 to S.-M.H.), Strategic Priority Research Program of CAS (XDB13040600 to Y.F.), NCMIS CAS and municipal government of Beijing.
Integrated supplementary information
Disulfide bonds identified from the E. coli periplasmic fraction
Potential substrates of DsbA
Potential substrates of DsbC
Disulfide bonds identified in human A549 cells
Glutathionylation sites identified in human A549 cells
Disulfide bonds identified in secreted HUVEC proteins
Candidate allosteric disulfide bonds in HUVEC proteins