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

Published

Author(s)

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

Abstract

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.
Citation
Journal of Proteome Research

Keywords

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

Citation

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 (Accessed September 17, 2021)
Created June 22, 2021