Human growth hormone proteoform pattern changes in pituitary adenomas: Potential biomarkers for 3P medical approaches

Abstract

Relevance

Human growth hormone (hGH) is synthesized, stored, and secreted by somatotroph cells in the pituitary gland, and promotes human growth and metabolism. Compared to a normal pituitary, a GH-secreting pituitary adenoma can secrete excessive GH to cause pathological changes in body tissues. GH proteoform changes would be associated with GH-related disease pathogenesis.

Purpose

This study aimed to elucidate changes in GH proteoforms between GH-secreting pituitary adenomas and control pituitaries for the predictive diagnostics, targeted prevention, and personalization of medical services.

Methods

The isoelectric point (pI) and relative molecular mass (Mr) are two basic features of a proteoform that can be used to effectively array and detect proteoforms with two-dimensional gel electrophoresis (2DGE) and 2DGE-based western blot. GH proteoforms were characterized with liquid chromatography (LC) and mass spectrometry (MS). Phosphoproteomics, ubiquitinomics, acetylomics, and bioinformatics were used to analyze post-translational modifications (PTMs) of GH proteoforms in GH-secreting pituitary adenoma tissues and control pituitaries.

Results

Sixty-six 2D gel spots were found to contain hGH, including 46 spots (46 GH proteoforms) in GH-secreting pituitary adenomas and 35 spots (35 GH proteoforms) in control pituitaries. Further, 35 GH proteoforms in control pituitary tissues were matched with 35 of 46 GH proteoforms in GH-secreting pituitary adenoma tissues; and 11 GH proteoforms were presented in only GH-secreting pituitary adenoma tissues but not in control pituitary tissues. The matched 35 GH proteoforms showed quantitative changes in GH-secreting pituitary adenomas compared to the controls. The quantitative levels of those 46 GH proteoforms in GH-secreting pituitary adenomas were significantly different from those 35 GH proteoforms in control pituitaries. Meanwhile, different types of PTMs were identified among those GH proteoforms. Phosphoproteomics identified phosphorylation at residues Ser77, Ser132, Ser134, Thr174, and Ser176 in hGH. Ubiquitinomics identified ubiquitination at residue Lys96 in hGH. Acetylomics identified acetylation at reside Lys171 in hGH. Deamination was identified at residue Asn178 in hGH.

Conclusion

These findings provide the first hGH proteoform pattern changes in GH-secreting pituitary adenoma tissues compared to control pituitary tissues, and the status of partial PTMs in hGH proteoforms. Those data provide in-depth insights into biological roles of hGH in GH-related diseases, and identify hGH proteoform pattern biomarkers for treatment of a GH-secreting pituitary adenoma in the context of 3P medicine –predictive diagnostics, targeted prevention, and personalization of medical services.

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Data availability

All data and materials are provided in this article and supplemental materials, which can be available publicly.

Code availability

All protein and gene accession codes can be available in the Swiss-Prot and Genbank databases. Bio-Rad PDQuest 2D gel image analysis software (version 7.0) is commercially available.

Abbreviations

ddH2O:

Deionized distilled water

DTT:

Dithiothreitol

ESI:

Electrospray ionization

hGH:

Human growth hormone

IEF:

Isoelectric focusing

IPG:

Immobilized pH gradient

LC:

Liquid chromatography

MALDI:

Matrix-assisted laser desorption ionization

MS:

Mass spectrometry

Mr:

Relative molecular mass

PBS:

Phosphate-buffered saline

pI:

Isoelectric point

PTMs:

Posttranslational modifications

PVDF:

Polyvinylidene fluoride

SDS-PAGE:

Sodium dodecyl sulfate–polyacrylamide gel electrophoresis

2DGE:

Two-dimensional gel electrophoresis

TOF:

Time-of-flight

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Acknowledgments

The authors acknowledge the financial support from the Shandong First Medical University Talent Introduction Funds (to X.Z.), the Hunan Provincial Hundred Talent Plan (to X.Z.), and the grants from China “863” Plan Project (Grant No. 2014AA020610-1 to XZ).

Funding

This work was supported by the Shandong First Medical University Talent Introduction Funds (to X.Z.), the Hunan Provincial Hundred Talent Plan (to X.Z.), and China “863” Plan Project (Grant No. 2014AA020610-1 to XZ).

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Authors

Contributions

B.L. analyzed data, carried out partial experiments, prepared figures and tables, and wrote the manuscript draft. X.W. performed 2DGE-based western blot experiment and mass spectrometry sample preparation. S.W., J.L., N.L., Y.L., and Y.M. participated in partial data analysis and experiment. J.L. assisted in 2DGE experiment and 2DGE image analysis. C.Y. provided control pituitary tissue samples and clinical diagnosis. Q.L. and X.L. obtained pituitary adenoma tissue samples and clinical diagnosis. D.M.D. provided control pituitary samples and critically reviewed the manuscript. X.Z. conceived the concept, designed experiments and manuscript, instructed experiments, analyzed data, obtained the ubiquitinated, phosphorylated, and acetylated proteomic data, supervised results, coordinated, wrote and critically revised manuscript, and was responsible for its financial supports and the corresponding works. All authors approved the final manuscript.

Corresponding author

Correspondence to Xianquan Zhan.

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The authors declare that there is no conflict of interests regarding the publication of this article.

Ethical approval

All the patients were informed about the purposes of the study and consequently have signed their “consent of the patient”. All investigations conformed to the principles outlined in the Declaration of Helsinki and were performed with permission (Approval number: 2013030181) by the responsible Medical Ethics Committee of Xiangya Hospital, Central South University, China.

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Supplementary information

Supplemental material provides additional information about the reactors and metagenome analysis discussed in the text and is available online on the Springer publications website at https://link.springer.com

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Li, B., Wang, X., Yang, C. et al. Human growth hormone proteoform pattern changes in pituitary adenomas: Potential biomarkers for 3P medical approaches. EPMA Journal 12, 67–89 (2021). https://doi.org/10.1007/s13167-021-00232-7

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Keywords

  • Growth hormone-secreting pituitary adenoma
  • Human growth hormone (hGH)
  • Two-dimensional gel electrophoresis (2DGE)
  • Liquid chromatography (LC)
  • Mass spectrometry (MS)
  • 2DGE-based western blot
  • Bioinformatics
  • Post-translational modifications (PTMs)
  • Phosphorylation
  • Acetylation
  • Ubiquitination
  • Phosphoproteomics
  • Acetylomics
  • Ubiquitinomics
  • Patient stratification
  • Prediction/prognostic assessment
  • Predictive, preventive, and personalized medicine (PPPM/3P medicine)