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. 2019 May 16;10:2195. doi: 10.1038/s41467-019-10182-4

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© The Author(s) 2019

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 license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license 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 license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 1 — Schematic representation of Cys-BOOST. The workflow is exemplified for SNO analysis of untreated (n = 3) and GSNO-treated (n = 3) HeLa cell extracts. Cys-BOOST incorporates the following steps: (1) irreversible blocking of all free thiols with IAA, (2) switch of specifically reduced Cys with alkyne-derivatized iodoacetamide reagent (IAA-alkyne), (3) reduction and alkylation of the remaining (endogenously) oxidized Cys, (4) protein digestion with trypsin, (5) TMT 10plex™ labeling of Lys residues and N-termini for multiplexed quantitative analysis, (6) conjugation of the Dde-biotin-azide linker to IAA-alkyne labeled peptides via copper(I)-catalyzed alkyne-azide cycloaddition (CuAAC), (7) affinity binding of the Cys peptides to streptavidin beads via the biotin group of the Dde-biotin-azide linker, (8) release of the target peptides with 2% hydrazine by single step mild chemical cleavage of the Dde bond, (9) on-tip pH 10 fractionation of the eluate before (10) LC-MS/MS analysis