A quantitative thiol reactivity profiling platform to analyze redox and electrophile reactive cysteine proteomes

Abstract

Cysteine is unique among all protein-coding amino acids, owing to its intrinsically high nucleophilicity. The cysteinyl thiol group can be covalently modified by a broad range of redox mechanisms or by various electrophiles derived from exogenous or endogenous sources. Measuring the response of protein cysteines to redox perturbation or electrophiles is critical for understanding the underlying mechanisms involved. Activity-based protein profiling based on thiol-reactive probes has been the method of choice for such analyses. We therefore adapted this approach and developed a new chemoproteomic platform, termed ‘QTRP’ (quantitative thiol reactivity profiling), that relies on the ability of a commercially available thiol-reactive probe IPM (2-iodo-N-(prop-2-yn-1-yl)acetamide) to covalently label, enrich and quantify the reactive cysteinome in cells and tissues. Here, we provide a detailed and updated workflow of QTRP that includes procedures for (i) labeling of the reactive cysteinome from cell or tissue samples (e.g., control versus treatment) with IPM, (ii) processing the protein samples into tryptic peptides and tagging the probe-modified peptides with isotopically labeled azido-biotin reagents containing a photo-cleavable linker via click chemistry reaction, (iii) capturing biotin-conjugated peptides with streptavidin beads, (iv) identifying and quantifying the photo-released peptides by mass spectrometry (MS)-based shotgun proteomics and (v) interpreting MS data by a streamlined informatic pipeline using a proteomics software, pFind 3, and an automatic post-processing algorithm. We also exemplified here how to use QTRP for mining H2O2-sensitive cysteines and for determining the intrinsic reactivity of cysteines in a complex proteome. We anticipate that this protocol should find broad applications in redox biology, chemical biology and the pharmaceutical industry. The protocol for sample preparation takes 3 d, whereas MS measurements and data analyses require 75 min and <30 min, respectively, per sample.

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Fig. 1: Methodologies for indirect profiling of redox and/or electrophile reactive cysteines.
Fig. 2: Overview of QTRP.
Fig. 3: Display of LC-MS/MS data from a representative .raw file.
Fig. 4: Representative MS1-based quantification results of typical QTRP experiments.

Data availability

The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE71 partner repository with the dataset identifier PXD016048. Software availability: post-processing algorithm (https://github.com/morpheusliu/Post-processing-program-for-pFind3-results).

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Acknowledgements

We thank M. Albertolle of The Scripps Research Institute and A. Foti of the Max Planck Institute for Infection Biology Cellular Microbiology for insightful comments. We thank K. S. Carroll of The Scripps Research Institute for kindly providing the S-sulfenic acid specific probe BTD. The work was supported by grants from the National Key R&D Program of China (2016YFA0501303 to J.Y.), the Natural Science Foundation of China (21922702, 81973279 and 31770885 to J.Y. and 31800036 to L.F.) and the State Key Laboratory of Proteomics (SKLP-K201703 and SKLP-K201804 to J.Y.). We also thank B. Fu and B. Zhong from the MS facility of the National Center for Protein Sciences • Beijing for their help and technical support.

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Authors

Contributions

L.F. and J.Y. developed the QTRP protocol and designed the studies. L.F., Z.L. C.T., Jixiang He and Jingyang He performed the experiments and analyzed the data. K.L. developed the post-processing algorithm. F.H., P.X. and J.Y. supervised the project. J.Y. wrote the manuscript.

Corresponding author

Correspondence to Jing Yang.

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The authors declare no competing interests.

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Peer review information Nature Protocols thanks Wei-Jun Qian, Johannes Herrmann and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Related links

Key references using this protocol

Fu, L. et al. Mol. Cell. Proteomics 16, 1815–1828 (2017): https://www.mcponline.org/content/16/10/1815.long

Sun, R. et al. Chem. Res. Toxicol. 30, 1797–1803 (2017): https://pubs.acs.org/doi/10.1021/acs.chemrestox.7b00183

Petrova, B. et al. Proc. Natl Acad. Sci. USA 115, E7978–E7986 (2018): http://www.pnas.org/content/115/34/E7978

Pei, J.-F. et al. Nat. Cell Biol. 21, 1553–1564 (2019): https://www.nature.com/articles/s41556-019-0420-4

Wang, W. et al. Cell Host Microbe 27, 601–613 (2020): https://www.sciencedirect.com/science/article/abs/pii/S1931312820301682

Supplementary information

Supplementary Information

Supplementary Figs. 1–13.

Reporting Summary

Supplementary Data 1

Proteome-wide mapping of oxidation-sensitive cysteines in HEK293T cells by QTRP. HEK293T cell lysates stimulated with or without H2O2 (1 mM, 15 min, 37 °C) and labeled with 100 mM IPM. The IPM-labeled proteome was then processed into tryptic peptides. The resulting probe-labeled peptides were conjugated with both L and H azido-UV-cleavable-biotin (Az-UV-biotin) reagents (1:1) via CuAAC. The L and H ‘Click’ reaction mixtures were cleaned with or without SCX. The biotinylated peptides were captured with streptavidin and photoreleased for LC-MS/MS–based identification and quantification. High RH/L values are indicative of cysteines with less free thiol being available after H2O2 treatment, suggesting potential redox-sensitive targets. A schematic workflow is shown in Fig. 2a.

Supplementary Data 2

Quantitative profiling of the intrinsic reactivity of proteomic cysteines. HEK293T proteomes were labeled with low (10 µM) and high (100 µM) levels of IPM. The probe-labeled proteins were processed into tryptic peptides. The resulting probe-labeled peptides were conjugated with both L and H azido-UV-cleavable-biotin (Az-UV-biotin) reagents (1:1) via CuAAC. The light and heavy ‘Click’ reaction mixtures were cleaned with or without SCX. The biotinylated peptides were captured with streptavidin and photoreleased for LC-MS/MS–based identification and quantification. Those with a calculated ratio of H to L (100 µM versus 10 µM, R10:1) <2.0 were defined as hyperreactive. A schematic workflow is shown in Fig. 2b.

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Fu, L., Li, Z., Liu, K. et al. A quantitative thiol reactivity profiling platform to analyze redox and electrophile reactive cysteine proteomes. Nat Protoc (2020). https://doi.org/10.1038/s41596-020-0352-2

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