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Comprehensive Analysis of Tryptic Peptides Arising from Disulfide Linkages in NISTmAb and Their Use for Developing a Mass Spectral Library



Qian Dong, Xinjian Yan, Yuxue Liang, Sanford Markey, Sergey L. Sheetlin, Concepcion Remoroza, William E. Wallace, Stephen Stein


This work presents methods for identifying and then creating a mass spectral library for disulfide-linked peptides originating from the NISTmAb. Analysis involved both partially- reduced and non-reduced proteins under neutral and weakly basic conditions, followed by nanoflow liquid chromatography tandem mass spectrometry (LC-MS/MS). This led to the detection of 428 distinct disulfide-linked peptide ions, arising from fully-tryptic cleavages, missed cleavages, irregular cleavages, complex Met/Trp oxidation mixtures, and metal adducts. Fragmentation features of disulfide bonds under low energy collision dissociation were examined. These include: (1) peptide bond cleavage leaving disulfide bonds intact; (2) disulfide bond cleavage, often leading to extensive fragmentation; and (3) double cleavage products resulting from both peptide and disulfide cleavages. Detailed annotation of various complex MS/MS fragments enabled the identification of disulfide-linked peptides with high confidence. Peptides containing each of the eight native disulfide bonds were identified along with 55 additional disulfide linkages arising from S-S bond shuffling. The presence of shuffled disulfides was nearly completely abrogated by refining digest conditions. A curated spectral library of 702 S-S linked peptide spectra was derived from this analysis and is available for download. Since all IgG1 antibodies have the same constant regions, the resulting library can be used as a tool for facile identification of disulfide-bonded peptides. Moreover, we show that one may identify such peptides originating from IgG1 proteins in human serum, thereby serving as a means of monitoring the completeness of protein reduction in many proteomics studies.
Journal of Proteome Research


disulfide-linked peptides, Disulfide bond connectivity, NISTmAb reference material, IgG1 mAb, MS spectral library, full MS scan, HCD fragmentation, high resolution LC-MS/MS


Dong, Q. , Yan, X. , Liang, Y. , Markey, S. , Sheetlin, S. , Remoroza, C. , Wallace, W. and Stein, S. (2021), Comprehensive Analysis of Tryptic Peptides Arising from Disulfide Linkages in NISTmAb and Their Use for Developing a Mass Spectral Library, Journal of Proteome Research, [online], (Accessed June 17, 2024)


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Created June 22, 2021, Updated October 14, 2021