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Impact of phosphorylation on thermal stability of proteins

Nature Methods volume 18, pages 757–759 (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)

Reversible protein phosphorylation regulates virtually every cellular process and is arguably the most well-studied post-translational modification. Still, less than 3% of phosphorylation sites identified in humans have annotated functions1; thus, the characterization of phosphorylation-site function is of high biological interest. Recently, Huang et al. introduced a proteomic method called Hotspot Thermal Profiling to detect effects of phosphorylation on the thermal stability of proteins in live cells2. We agree with the premise of the Huang et al. study that phosphorylation sites that have a significant effect on protein thermal stability are more likely to be functionally relevant, for example, by modulating protein conformation, localization and protein interactions3 (Supplementary Discussion, ‘Interpreting thermal stability shifts’). However, we have carefully examined their data and designed an optimized protocol combining thermal proteome profiling (TPP)4 with phosphoproteomics (phospho–TPP), which leads us to conclude that the impact of phosphorylation on protein thermal stability is likely to be less extensive than Huang et al. originally reported.

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Fig. 1: Global impact of phosphorylation on thermal stability of proteins.

Data availability

Mass spectrometry proteomic data were deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD015993.

Code availability

All code to reproduce the analysis and figures is available at https://github.com/nkurzaw/phosphoTPP.

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Acknowledgements

This work was supported by the European Molecular Biology Laboratory. C.M.P. and A.M. were supported by a fellowship from the EMBL Interdisciplinary Postdoctoral (EI3POD) Programme under Marie Skłodowska-Curie Actions COFUND (grant number 664726). N.K. was supported by a fellowship from the EMBL International PhD Programme.

Author information

Author notes

  1. These authors contributed equally: Clément M. Potel, Nils Kurzawa, Isabelle Becher.

Authors and Affiliations

  1. Genome Biology Unit, European Molecular Biology Laboratory, Heidelberg, Germany

    Clément M. Potel, Nils Kurzawa, Isabelle Becher, Athanasios Typas, André Mateus & Mikhail M. Savitski

  2. Faculty of Biosciences, Heidelberg University, Heidelberg, Germany

    Nils Kurzawa

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Contributions

C.M.P., I.B. and A.M. performed experimental work. N.K. performed data analysis. A.M. and M.M.S. supervised the work. All authors contributed to interpreting data and writing the manuscript.

Corresponding authors

Correspondence to André Mateus or Mikhail M. Savitski.

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The authors declare no competing interests.

Supplementary information

Supplementary Information

Supplementary Discussion, Figs. 1–11 and Table 1

Reporting Summary

Supplementary Data 1

Phosphopeptide fold changes at each temperature in each replicate (gene_name, gene name; protein_id, UniProt ID; sequence, peptide sequence; mod_sequence, modified peptide sequence; phospho_site_STY, number of modified amino acids in the peptide sequence; rel_fc_TMT_REP_TEMP, relative fold changes to 37 °C (TMT, TMT label; REP, replicate; TEMP, temperature).

Supplementary Data 2

Unmodified peptide fold changes at each temperature in each replicate (gene_name, gene name; protein_id, UniProt ID; sequence, peptide sequence; modifications, peptide modifications; rel_fc_TMT_REP_TEMP, relative fold changes to 37 °C (TMT, TMT label; REP, replicate; TEMP, temperature).

Supplementary Data 3

Comparison of melting behavior of phosphopeptides and unmodified peptides (gene_name, gene name; Gene_pSite, phosphosite position within protein; mod_sequence, modified peptide sequence; Tm_mean_phospho, average melting temperature of phosphopeptide; Tm_mean_unmodified, average melting temperature of unmodified protein; mean_delta_meltPoint, average difference of melting temperature of phosphopeptide and unmodified protein; significant, significantly different melting temperature of phosphopeptide compared to unmodified protein; functional_score, predicted functional importance of phosphosite position by aggregating multiple parameters for each phosphosite using machine learning and ranges from 0 to 1, with a higher value representing a higher probability that the phosphosite has functional relevance; Tm_phospho_REP, melting temperature of phosphopeptide (REP, replicate); Tm_unmodified_REP, melting temperature of unmodified protein; delta_meltPoint_REP, difference of melting temperature of phosphopeptide and unmodified protein; p_adj_REP, P value adjusted for multiple-testing correction; phospho_median_fc_TEMP, median relative fold changes to 37 °C of phosphopeptide (TEMP, temperature); unmodified_median_fc_TEMP, median relative fold changes to 37 °C of unmodified protein.

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Potel, C.M., Kurzawa, N., Becher, I. et al. Impact of phosphorylation on thermal stability of proteins. Nat Methods 18, 757–759 (2021). https://doi.org/10.1038/s41592-021-01177-5

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