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Identification of phosphosites that alter protein thermal stability

Nature Methods volume 18, pages 760–762 (2021)Cite this article

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Matters Arising to this article was published on 17 June 2021

The Original Article was published on 05 August 2019

arising from J. X. Huang et al. Nature Methods https://doi.org/10.1038/s41592-019-0499-3 (2019)

Proteomic methods have enabled the discovery of hundreds of thousands of protein phosphorylation sites1; however, we lack methods to systematically annotate their function. Phosphorylation has numerous biological functions that biochemically involve changes in protein structure and interactions. These biochemical changes can be detected by measuring the difference in thermal stability between the protein and the phosphoprotein. Recently, Huang et al.2 developed the Hotspot Thermal Profiling (HTP) method to identify phosphosites that alter protein thermal stability, reporting 719 of 2,883 (25%) measured human phosphoisoforms from HEK293T cells with significant effects.

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Fig. 1: Analysis of phosphosite effects on protein stability.
Fig. 2: Examples of phosphosites that alter protein thermal stability.

Data availability

The MS proteomic data generated for this study were deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD016750.

Code availability

All code to reproduce analysis and data figures is available at https://gitlab.com/public_villenlab/dali_phospho_thermalstability.

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Acknowledgements

We thank members of the Villén laboratory for scientific discussions, in particular, B. Ruiz, M. Leutert and A. Hogrebe. We thank A. Llovet Soto and J. Eng for software developments on the data analysis pipeline. I.R.S. and K.N.H. were supported by NIH training grant T32HG000035. A.S.V. was supported by NIH training grant T32LM012419. Most of this work was supported by NIH grant R35GM119536 to J.V. The Villén laboratory is additionally supported by NIH grants R01AG056359, R01NS098329 and RM1HG010461; Human Frontiers Science Program grant RGP0034/2018; a research program grant from the WM Keck Foundation; and the University of Washington Proteome Resource UWPR95794.

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Authors and Affiliations

  1. Department of Genome Sciences, University of Washington, Seattle, WA, USA

    Ian R. Smith, Kyle N. Hess, Anna A. Bakhtina, Anthony S. Valente, Ricard A. Rodríguez-Mias & Judit Villén

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  1. Ian R. Smith
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Contributions

I.R.S., K.N.H., R.A.R.-M. and J.V. conceived the study and designed experiments. I.R.S. conducted experiments with advice from K.N.H., R.A.R.-M. and J.V. and assistance from A.A.B. I.R.S. analyzed data with advice from R.A.R.-M. and A.S.V. J.V. supervised the study. I.R.S. and J.V. wrote the paper, and all authors edited it.

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Correspondence to Judit Villén.

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Extended data

Extended Data Fig. 1 Reproducibility and robustness of Dali compared to HTP.

a, Scatter plot and Pearson correlation between the mean Tm for unmodified peptides observed in the phosphopeptide enriched samples (n = 10) and the mean Tm for their corresponding proteins (n = 11). Results from the Huang et al. data reanalysis conducted by us. b, Scatter plot and Pearson correlation as in a with Rs values obtained from the Dali method (n = 6).

Extended Data Fig. 2 Phosphosites that significantly alter protein thermal stability using two different statistical settings.

Volcano plots showing ΔTm for mean phosphopeptide isoform to mean protein counterpart in the x axis, and the two-sided Student’s t-test probability in the y axis. a, Huang et al. implementation shows a P value because multiple hypothesis correction was not applied. Significant phosphopeptide isoforms (blue) are defined by P value < 0.05. b, Our proposed analysis consolidates data from MS reanalysis prior to statistical testing using a two-sided Welch’s t-test, which is performed assuming unequal variances between phosphopeptide isoform and proteins. Benjamini-Hochberg adjustment was used to correct P values for multiple hypothesis testing. Significant phosphopeptide isoforms (blue) are defined by q value < 0.05.

Extended Data Fig. 3 Examples of significant hits on proteins that undergo posttranslational splicing or cleavage.

a, Rs values for observed VMA1 unmodified peptides identified in phosphopeptide-enriched samples and proteome samples displayed across the length of VMA1. Spliced products from amino acid 2–283 and 738–1031 are joined to generate the V-type proton ATPase catalytic subunit A proteoform, extinguishing the 284–737 segment. Peptides derived from the proteome samples are colored in gray and significant unmodified peptides found in the phosphopeptide-enriched sample are in red. b, Similar plot to a for RPS31, which is cleaved to generate ubiquitin (1–76 amino acid segment) and 40 S ribosomal protein S31 (77–152 amino acid segment) proteins.

Extended Data Fig. 4 Examples of phosphosites that alter protein thermal stability and are located at protein interfaces.

Rs boxplots for a, ARO8 S59, b, TPI1 S79, and c, GAPDH S201 phosphopeptide isoforms and their protein counterparts. All boxplots show results from n = 6 biological replicates, and the line represents the median, the box designates the interquartile range (IQR), and the whiskers define 1.5*IQR from the box ends. ARO8 S59, TPI1 S79, and GAPDH S201 reside at dimerization interfaces as shown in the structures to the right (PDB accession: 4JE5, 1NEY, and 3PYM, respectively). Phosphomimetic mutations ARO8 S59E and TPI1 S79E are predicted to disrupt protein interfaces (ΔΔGpred = 3.78 and ΔΔGpred =8.04 respectively). Additionally, TPI1 S79E mutation is predicted to alter protein conformational stability (ΔΔGpred = 2.39). ΔΔGpred > 2 is predicted to be destabilizing.

Extended Data Fig. 5 Examples of phosphosites that alter protein thermal stability on glycolytic enzymes.

a, Rs values for PGK1 and all measured PGK1 phosphopeptide isoforms, with significantly destabilizing phosphosite S331 shown in red. ΔΔGpred for all glutamic acid phosphomimetic substitutions were obtained from mutfunc, with ΔΔGpred > 2 considered likely destabilizing. b, GAPDH S149 phosphopeptide is shared across all GAPDH paralogs (TDH1, TDH2, and TDH3). Boxplot shows Rs values and distributions for peptides unique to one isoform (TDH1, TDH2, TDH3), peptides shared among all GAPDH isoforms (all), all peptides for TDH3, and the S149 phosphopeptide isoform. Bottom panel shows localization of S149 on the GAPDH structure near the binding site of the enzyme substrate. Boxplots show results from 6 biological replicates, the line represents the median, the box designates the interquartile range (IQR), and the whiskers define 1.5*IQR from the box ends.

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Smith, I.R., Hess, K.N., Bakhtina, A.A. et al. Identification of phosphosites that alter protein thermal stability. Nat Methods 18, 760–762 (2021). https://doi.org/10.1038/s41592-021-01178-4

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