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Histidine phosphorylation in human cells; a needle or phantom in the haystack?


It has been suggested that in mammalian cells histidine residues in proteins may become as frequently phosphorylated as serine, threonine and tyrosine, and may play a key role in mammalian signaling. Here we applied a robust workflow that earlier allowed us to detect histidine phosphorylation in bacteria unambiguously, to probe for histidine phosphorylation in four human cell lines. Initially, seemingly hundreds of protein histidine phosphorylations were picked up in all studied human cell lines. However, careful examination of the data, and several control experiments, led us to the conclusion that >99% of these initially assigned pHis sites were not genuine, and should be site localized to neighboring Ser/Thr residues. Nevertheless, our methods are selective enough to detect just a handful of genuine pHis sites in mammalian cells, representing well-known enzymatic intermediates. Consequently, we do not find any evidence in our data supporting that protein histidine phosphorylation plays a role in mammalian signaling.

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Fig. 1: Initially widespread detection of pHis peptides in tryptic digests of human cells reduces to just a handful of genuine ones after stringent filtering.

Data availability

All raw data that support the findings of this study have been deposited in ProteomeXchange with the accession number PXD028360.


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We thank B. van Breukelen for assistance with preparing the figures. This research received funding through the Netherlands Organization for Scientific Research through a VIDI grant (project no. 723.013.008) to S.L. and the Spinoza Award SPI.2017.028 to A.J.R.H. This project received additional funding from the European Union’s Horizon 2020 research and innovation program under grant agreement no. 686547 (EPIC-XS).

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N.M.L., A.J.R.H. and S.L. designed the experiments. N.M.L. performed the experiments. N.M.L. and S.L. performed data analysis. N.M.L., A.J.R.H. and S.L. wrote the manuscript.

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Correspondence to Simone Lemeer.

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Nature Methods thanks the anonymous reviewers for their contribution to the peer review of this work. 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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Supplementary Notes 1–6 and Supplementary Figs. 1–7

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Leijten, N.M., Heck, A.J.R. & Lemeer, S. Histidine phosphorylation in human cells; a needle or phantom in the haystack?. Nat Methods 19, 827–828 (2022).

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