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. 2021 Oct 11;11:20131. doi: 10.1038/s41598-021-99696-w

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© The Author(s) 2021

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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Production of DIII blocking peptides and assessment of the presence of disulfide bond. (A) Amplicons encoding tagged peptides resolved on the agarose gel; (B) Purified tagged peptides on LDS-PAGE. (C) Molecular mass of the representative cyclic peptide (CP2) confirmed by MALDI-TOF MS; (D) Molecular mass of the representative linear peptide (LP3) confirmed by MALDI-TOF MS. Predicted masses of tagged peptides (~ 7 kDa) corresponded to observed masses in MALDI-TOF MS (C and D), however, tagged peptides had slower migration in LDS-PAGE (B). In (C and D): I – tagged peptide; II – the tag (6xHis tag – 28 aa tag – GGGGS linker) after enterokinase digestion; III – purified peptide after removal of the tag. (E) The presence of disulfide bonds in cyclic peptides was confirmed. Any free thiols present in the peptides were blocked with N-ethylmaleimid (NEM). Peptides were then either reduced (R) or maintained in oxidized form (O). Both R and O forms were incubated with thiol-reactive IRDye 800CW Maleimide and then separated on non-reducing LDS-PAGE. In the peptide, if thiols are occupied with the disulfide bond, they remain unblocked and get reduced with TCEP. Free thiols in reduced peptides are then labeled giving a green signal. In the oxidized form, no free thiols are available, thus no labeling occurs (no green signal).