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Mass spectrometry and enzyme assays refute histone tyrosine sulfation

Nature Chemical Biology (2025)Cite this article

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Matters Arising to this article was published on 01 September 2025

The Original Article was published on 20 February 2023

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arising from W. Yu et al. Nature Chemical Biology https://doi.org/10.1038/s41589-023-01267-9 (2023)

Tyrosylprotein sulfotransferases are only known to exist in the Golgi, consistent with tyrosine sulfation occurring in secretory and membrane proteins1. Thus, the findings of Yu et al.2 that histone H3 sulfation occurs in the cytosol are surprising. Cytosolic sulfotransferase SULT1B1, which sulfates phenolic small molecules, was proposed to catalyze H3Y99 sulfation with 3′-phosphoadenosine 5′-phosphosulfate (PAPS) as SO3 donor. Bottom–up nanoflow liquid chromatography–tandem mass spectrometry (nanoLC–MS/MS)-based proteomic analyses were key to this conclusion. Such experiments appear to have been performed following standard protocols, including reduction (reagent unknown), alkylation with iodoacetamide, and digestion prior to separation of proteolytic peptides and data-dependent higher-energy collision dissociation (HCD) to generate sequence-informative peptide fragments. However, the published HCD MS/MS spectra do not match established sulfopeptide fragmentation behavior. We present reannotation of these spectra and additional experiments that found a lack of evidence to support H3Y99 sulfation.

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Fig. 1: H3Y99 sulfation is misannotated in Yu et al. HCD MS/MS spectra.
Fig. 2: Lack of LC–MS/MS evidence supporting histone H3.2. Y99 sulfation upon incubation with SULT1B1 and PAPS.

Data availability

All MS data are available in MassIVE under identifier MSV000095851. Source data are provided with this paper.

References

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Acknowledgements

This work was supported by NIH grant no. R01GM139916 and an Agilent Thought Leader award to K.H. M.M.Y. acknowledges support from the Ministry of Higher Education of the Arab Republic of Egypt.

Author information

Authors and Affiliations

  1. Department of Chemistry, University of Michigan, Ann Arbor, MI, USA

    Menatallah M. Youssef, Carson W. Szot, Ryan C. Bailey & Kristina Hakansson

  2. Department of Pharmaceutical Analytical Chemistry, Faculty of Pharmacy, Ain Shams University, Cairo, Egypt

    Menatallah M. Youssef, Miriam F. Ayad, Lobna A. Hussein & Maha F. Abdel-Ghany

  3. Department of Pathology, University of Michigan Medical School, Ann Arbor, MI, USA

    Ahmed M. Mostafa

  4. Department of Biochemistry, Faculty of Pharmacy, Ain Shams University, Cairo, Egypt

    Ahmed M. Mostafa

  5. Ion Cyclotron Resonance Program, National High Magnetic Field Laboratory and Department of Chemistry & Biochemistry, Florida State University, Tallahassee, FL, USA

    Kristina Hakansson

Authors
  1. Menatallah M. Youssef
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Contributions

M.M.Y., C.W.S., A.M.M. and K.H. performed methodology and experiments. M.M.Y., R.C.B. and A.M.M. carried out data analysis. M.M.Y., R.C.B. and K.H. wrote and edited the paper. M.F.A., L.A.H., M.F.A.-G. and K.H. carried out project administration.

Corresponding author

Correspondence to Kristina Hakansson.

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The authors declare no competing interests.

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Peer review information

Nature Chemical Biology thanks the anonymous reviewers for their contribution to the peer review of this work.

Extended data

Extended Data Fig. 1 Evidence of H3Y99 phosphopeptide presence in Yu et al. HepG2 nuclear extract, mixed and synthetic sulfopeptide samples.

Extracted ion chromatograms of the tyrosine 99 phosphopeptide 3+ precursor ion (706.6709 m/z), at 6 ppm mass tolerance, in the HepG2 nuclear extract sample (a), mixed sample (b) and synthetic sulfopeptide sample (c) from Yu et al. The phosphopeptide was observed eluting at 14.09-14.18 min. in those short 30 min. gradient proteomic analysis runs. The corresponding MS1 spectrum of the H3Y99 phosphopeptide precursor, eluting at 14.09 min in the HepG2 nuclear extract sample, showed 1.56e7 abundance (inset in a) compared to an abundance of 2.56e3 at 25.45 min (d) proposed by Yu et al. MS2 spectrum supporting phosphopetide assignment was also absent around 25.45 min in Yu et al. HepG2 sample. Moreover, no evidence of phosphopeptide was observed around this retention time in both Yu et al. mixed and synthetic sulfopeptide samples.

Extended Data Fig. 2 H3Y99 sulfation is misannotated in HCD MS/MS spectra from Yu et al.’s short proteomic runs.

AyLVGLFEDTNLC(carbamidomethyl)AIHAK, (y= sulfotyrosine) HCD MS/MS spectra in the HepG2 nuclear extract sample (a), mixed sample (b) and synthetic sulfopeptide sample (c) as shown by Yu et al. The HCD MS/MS spectra annotations suggest b-type ions retaining the SO3. Upon revisiting Yu et al.’s raw data, HCD MS/MS spectra showing a sulfopeptide fragmentation behavior with the complete SO3 neutral loss were observed in the HepG2 nuclear extract sample (d), mixed sample (e) and synthetic sulfopeptide sample (f).

Extended Data Fig. 3 Undetected Histone H3 Tyrosine Sulfation in HepG2 Lysates.

Western blot analyses were conducted with HepG2 whole cell lysate (WCL, ab166833) and nuclear extract lysate (NEL, ab14660) to detect tyrosine sulfation using an anti-sulfo-Y antibody (Abcam, ab136481; Bottom). Bovine fibrinogen (Fib., Sigma F8630, 0.5 µg/µl) and normal mouse liver tissue lysate were used as positive controls. β-actin (Proteintech, 66009) served as an internal control (Top). Anti-H3K27Me3 (Cell Signaling, 9733S) confirmed histone H3 presence (Middle). As expected, H3K27Me3 bands were more prominent in NEL compared to WCL, owing to nuclear histone localization. No sulfo-Y bands were observed at 15 kDa in either WCL or NEL, indicating insignificant histone tyrosine sulfation in HepG2 cells, with a faint band detected ~40 kDa only in NEL.

Source data

Extended Data Fig. 4 LC/Fourier transform ion cyclotron resonance (FT-ICR) MS detection of sulfated T3, the known SULT1B1 substrate.

Activity of rhSULT1B1 was optimized using its known substrate, T3, and LC/FT-ICR MS analysis achieved via positive polarity mode. Extracted ion chromatograms of sulfated T3 (a) showed an abundance of 8e8, while non-sulfated T3 (b) was at 2.5e9 abundance after incubation with rhSULT1B1 and PAPS at 37 °C overnight. Please note that sulfation reduces ionization efficiency in positive ion mode and, thus, the relative abundance in the chromatogram is not representative of actual concentrations.

Supplementary information

Reporting Summary

Supplementary Tables

Supplementary Table 1: AYLVGLFEDTNLC(carbamidomethyl)AIHAK tyrosine 99 sulfated and phosphorylated peptide fragment ions. Supplementary Table 2: Peptide groups from synthesized H3Y99-sulfopeptide Fig. 1d in Yu et al. nanoLC–HCD/MS/MS data. Supplementary Table 3: Protein groups from synthesized H3Y99-sulfopeptide Fig. 1d in Yu et al. nanoLC–HCD/MS/MS data. Supplementary Table 4: Peptide groups from synthesized H3Y99-sulfopeptide Fig. 1e in Yu et al. nanoLC–HCD/MS/MS data. Supplementary Table 5: Protein groups from synthesized H3Y99-sulfopeptide Fig. 1e in Yu et al. nanoLC–HCD/MS/MS data.

Source data

Source Data Extended Data Fig. 3

Unprocessed western blot.

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Youssef, M.M., Szot, C.W., Ayad, M.F. et al. Mass spectrometry and enzyme assays refute histone tyrosine sulfation. Nat Chem Biol (2025). https://doi.org/10.1038/s41589-025-01994-1

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