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Global profiling of phosphorylation-dependent changes in cysteine reactivity

Nature Methods volume 19, pages 341–352 (2022)Cite this article

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Abstract

Proteomics has revealed that the ~20,000 human genes engender a far greater number of proteins, or proteoforms, that are diversified in large part by post-translational modifications (PTMs). How such PTMs affect protein structure and function is an active area of research but remains technically challenging to assess on a proteome-wide scale. Here, we describe a chemical proteomic method to quantitatively relate serine/threonine phosphorylation to changes in the reactivity of cysteine residues, a parameter that can affect the potential for cysteines to be post-translationally modified or engaged by covalent drugs. Leveraging the extensive high-stoichiometry phosphorylation occurring in mitotic cells, we discover numerous cysteines that exhibit phosphorylation-dependent changes in reactivity on diverse proteins enriched in cell cycle regulatory pathways. The discovery of bidirectional changes in cysteine reactivity often occurring in proximity to serine/threonine phosphorylation events points to the broad impact of phosphorylation on the chemical reactivity of proteins and the future potential to create small-molecule probes that differentially target proteoforms with PTMs.

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Fig. 1: Cysteine reactivity profiling of mitotic and asynchronous cells.
Fig. 2: Proteomic mapping of phosphorylation-dependent changes in cysteine reactivity.
Fig. 3: Adapted protocol for interpreting proximal phosphorylation-cysteine interactions.
Fig. 4: Features of proteins with mitotic phosphorylation-dependent changes in cysteine reactivity.

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Data availability

All mass spectrometry data are available via PRIDE with identifier PXD026730. Source data, in addition to Supplementary Dataset 1, is available for all figure panels. Source data are provided with this paper.

Code availability

TMT-based data output from Integrated Proteomics Pipeline (IP2) and isoTOP data output from CIMAGE was further analyzed with custom scripts, available on Zenodo at https://zenodo.org/badge/latestdoi/419072418.

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Acknowledgements

We thank V. Vartabedian from the Teijaro lab for technical assistance and G. Simon (Vividion) and S. Niessen and M. Hayward (Pfizer) for valuable feedback throughout this project. This work was supported by the National Institutes of Health (NIH) (CA231991 to B. F. C), NIH-NCI (CA239556 to E. K. K.), and Vividion Therapeutics.

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  1. The Department of Chemistry and The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, CA, USA

    Esther K. Kemper, Yuanjin Zhang, Melissa M. Dix & Benjamin F. Cravatt

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  1. Esther K. Kemper
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Contributions

B. F. C. and E. K. K. conceived of the project, analyzed data, and wrote the manuscript. E. K. K. and M. M. D. developed mass spectrometry methods. E. K. K. and Y. Z. performed experiments.

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Correspondence to Esther K. Kemper or Benjamin F. Cravatt.

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Nature Methods thanks Dustin Maly and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Editor recognition statement Arunima Singh was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Extended data

Extended Data Fig. 1 Cysteine reactivity profiling of mitotic and asynchronous cells.

a, Timeline for mitotic HeLa cell proteome generation. b, Cysteine reactivity values for GAPDH and PARK7 (Mitosis/Asynch) cell proteomes. Horizontal black line for each cysteine marks median value, boxes mark the upper and lower quartiles, and whiskers mark 1.5x interquartile range for n = 9 independent experiments (circles). Dotted lines designate boundaries for ≥ twofold changes. c, TMT-ABPP workflow for measuring protein expression (top, blue) and cysteine reactivity (bottom, gray) in the mitotic and asynchronous HeLa cell proteome. d, Venn diagram showing overlap (light blue) in proteins quantified by TMT-ABPP (gray) and unenriched proteomics (dark blue). For inclusion, proteins had at least one quantified cysteine in at least two replicate experiments of TMT-ABPP and/or two unique quantified peptides quantified from at least one replicate of unenriched proteomics. e, GO cellular analysis of proteins with reactivity-based cysteine changes in mitotic vs asynchronous cell proteomes45,46. Proteins with reactivity-based cysteine changes correspond to those defined in Fig. 1d. f, Box plot showing DTYMK cysteine reactivity values (Mitosis/Asynch). Horizontal black line for each cysteine marks median value, boxes mark the upper and lower quartiles, and whiskers mark 1.5x interquartile range for n = 5 (or more) independent experiments (circles). Dotted lines designate boundaries for ≥ twofold changes. g, Cysteine reactivity values for all quantified DTYMK cysteines following gel filtration of the mitotic cell proteome. Data represent average values + /- standard deviation for n = 3 independent experiments (circles). h, DTYMK C117 reactivity following gel filtration of asynchronous (gray) vs mitotic (blue) cell proteomes. Data represent average values + /- standard deviation for n = 3 (or more) independent experiments (circles). i, X-ray crystal structure of DTYMK in complex with AMP and TMP with C163 and C117 highlighted in yellow (PDB: 1E2D)47. j, Protein expression values for DTWD1 (not detected), NOL8, and RRP15 (Mitosis/Asynch). Horizontal black line for each cysteine marks median value, boxes mark the upper and lower quartiles, and whiskers mark 1.5x interquartile range for n = 9 independent experiments (circles). Dotted lines designate boundaries for ≥ twofold changes.

Source data

Extended Data Fig. 2 A proteomic method to map phosphorylation-dependent changes in cysteine reactivity.

a, Venn diagram of phosphorylated S/T residues quantified by Sharma et al.21 (red) and this study (dark blue) in asynchronous and mitotic cell proteomes. b, Cysteine reactivity ratio values in Native/Denatured mitotic proteome. Light blue and orange data mark cysteine reactivity values that are ≥ twofold higher (boundary marked by dotted lines) in native and denatured cell proteome, respectively. Data are the median value for n = 1 (or more) independent experiments. c, Comparison of cysteine reactivity values from LPP(-)/LPP( + ) (y-axis) and Native/Denatured (x-axis) proteomes. Blue and red data mark cysteine reactivity values that are ≥ twofold higher in LPP(-) and LPP( + ) cell proteomes, respectively. Light blue and orange data mark cysteines that are unchanging in LPP(-)/LPP( + ), but changing twofold in Native/Denatured mitotic proteomes. Dotted lines mark boundaries for cysteines that change ≥ two-fold in reactivity in LPP(-)/LPP( + ) and Native/Denatured. Data are the median value for n = 2 (or more) independent LPP(-)/LPP( + ) experiments and n = 1 (or more) Native/Denatured experiments. d-f, Cysteine reactivity values across Native/Denatured proteome (orange) and LPP(-)/LPP( + ) (green) mitotic proteome for cysteines in d) FLNB, e) NUMA1, and f) BAG3. Horizontal black lines mark median value, boxes mark upper and lower quartiles, and whiskers mark 1.5x interquartile range for n = 2 (or more) independent experiments (circles). Dotted lines designate boundaries for ≥ twofold changes. g, Percentage of cysteines in predicted disordered domains (IUPreD ≥ 0.5)48. h, Cysteine reactivity values for MAP2K4 in gel-filtred mitotic cell proteome. Data represent the average values + /- standard deviation for n = 2 (or more) independent experiments (circles). i, MAP2K4 C246 reactivity in gel-filtered asynchronous (gray) vs mitotic (blue) cell proteomes. Data represent the average values + /- standard deviation for n = 4 (or more) independent experiments (circles). j, Left, MAP2K ATP-binding pocket cysteine reactivity. Non-unique peptides are assigned to both MAP2Ks. Horizontal black lines mark median value, boxes mark upper and lower quartiles, and whiskers mark 1.5x interquartile range for n = 2 (or more) independent experiments (circles). Dotted lines designate boundaries for ≥ two-fold changes. Right, sequence alignment of MAP2K proteins centered on MAP2K4 ATP-binding pocket C246.

Source data

Extended Data Fig. 3 Adapted protocol for interpreting proximal phosphorylation-cysteine interactions.

a, Left, cysteine reactivity values for indicated comparison groups for quantified cysteines from ECD3 (C137, top) and GTF2I (C215, bottom). Horizontal black line for each cysteine marks median value, boxes mark upper and lower quartiles, and whiskers mark 1.5x interquartile range for n = 5 (or more) independent experiments (circles). Dotted lines designate boundaries for ≥ two-fold changes. Right, tryptic peptides containing EDC3 C137 (asterisks, red, bold; top) and GTF2I C215 (asterisks, red, bold; bottom) and high occupancy phosphorylation sites (black, bold)21 b, Left, bar graph showing phosphopeptide enrichment of EDC3 p-S131 (top) and GTF2I p-S210 (bottom). Data were normalized to mitotic proteome without LPP treatment (Mitosis LPP(-)) and represent the median values ± standard deviation for n = 3 independent experiments (circles). Right, tryptic peptides containing phosphorylated (p-, purple, bold) EDC p-S131 (asterisks, purple, bold; top) and GTF2I p-S210 (asterisks, purple, bold; bottom) and cysteines from Extended Data Fig. 3a marked (red, bold). c, Immunoblot analysis of MAP2K1 with antibody #9146 in mitosis. Data are from a single experiment representative of two independent experiments. d, FLNA cysteine reactivity values across the indicated comparison groups. Horizontal black line for each cysteine marks median value, boxes mark the upper and lower quartiles, and whiskers mark 1.5x interquartile range for n = 5 (or more) independent experiments (circles). Dotted lines designate boundaries for ≥ two-fold changes. e, Left, SLAIN2 C152 reactivity values across the indicated comparison groups. Horizontal black line for each cysteine marks median value, boxes mark the upper and lower quartiles, and whiskers mark 1.5x interquartile range for n = 3 (or more) independent experiments (circles). Dotted lines designate boundaries for ≥ two-fold changes. Right, tryptic peptide containing SLAIN2 C152 (asterisks, blue, bold) and a potential S/T-P phosphorylation site (black, bold). f, Left, bar graph showing phosphopeptide enrichment of SLAIN2 p-S147. Data were normalized to mitotic proteome without LPP treatment (Mitosis LPP(-)) and are from n = 1 experiment. Right, tryptic peptide containing phosphorylated (p-, purple, bold) SLAIN2 S147 (asterisks, purple, bold) with cysteine from Extended Data Fig. 3e marked (blue, bold).

Source data

Extended Data Fig. 4 Features of proteins with mitotic phosphorylation-dependent changes in cysteine reactivity.

a, Proteins with authentic (left) and artifactual (middle) phosphorylation-dependent cysteine reactivity changes are enriched for high stoichiometry mitotic phosphorylation sites21 (blue) compared to all quantified proteins (right). Proteins lacking sufficient data for phosphorylation stoichiometry calculation or exhibiting only low stoichiometry (< 50% occupancy) sites21 were labeled as ‘Low or unquantified stoichiometry’ (orange). b, Proteins with phosphorylation-dependent cysteine reactivity changes (left) are enriched for LPP-sensitive mitotic phosphorylation sites (purple) compared to all quantified proteins (right). Proteins with only artifactual cysteine reactivity changes were removed from analysis. Proteins with LPP-insensitive or no quantified phosphorylation sites were labeled as “Unchanging or unquantified phosphosites” (gray). c, Members of the anaphase-promoting complex (APC/C) of the KEGG cell cycle pathway (HSA04110).55 Proteins are as described in Fig. 4b. d, Fraction of cysteines showing phosphorylation-dependent reactivity changes within the specified amino acid distances from an S/T-P site. Artifactual phosphorylation-dependent cysteine reactivity changes were omitted from analysis. e, Fraction of cysteines showing authentic (left) versus artifactual (right) phosphorylation-dependent reactivity changes within the specified amino acid distances from an S/T-P site. Authentic and artifactual changes were determined as described in Fig. 3f. f, Sequence alignment of the KLC1 and KLC2 proteins centered on C456 and C441, respectively (asterisks, red, bold). Known (KLC1) and predicted (KLC2) S-P phosphorylation motifs are marked (black, bold). g, KLC1 cysteine reactivity values across the indicated comparison groups. Horizontal black lines mark median value, boxes mark upper and lower quartiles, and whiskers mark 1.5x interquartile range for n = 2 (or more) independent experiments (circles). Dotted lines designate boundaries for ≥ two-fold changes. h, FXR2 C270 reactivity values for indicated comparison groups. Horizontal black lines mark median value, boxes mark upper and lower quartiles, and whiskers mark 1.5x interquartile range for n = 5 (or more) independent experiments (circles). Dotted lines designate boundaries for ≥ two-fold changes. i, Left, phosphopeptide enrichment of FXR2 p-S450. Data were normalized to Mitosis LPP(-) and represent the median values + /- standard deviation for n = 2 independent experiments (circles). Right, tryptic peptides containing phosphorylated (p-, purple, bold) FXR2 p-S450 (asterisks, purple, bold).

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Kemper, E.K., Zhang, Y., Dix, M.M. et al. Global profiling of phosphorylation-dependent changes in cysteine reactivity. Nat Methods 19, 341–352 (2022). https://doi.org/10.1038/s41592-022-01398-2

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