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Myoblast Phosphoproteomics as a Tool to Investigate Global Signaling Events During Myogenesis

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Myogenesis

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1889))

Abstract

Protein phosphorylation is a universal covalent chemical modification of amino acids involved in a large number of biological processes including cell signaling, metabolism, proliferation, differentiation, survival/death, ageing, and many more. Regulation of protein phosphorylation is essential in myogenesis and indeed, when the enzymatic activity of protein kinases is distrupted in myoblasts, myogenesis is affected. In this chapter we describe a method to profile the phosphoproteome of myoblasts using mass spectrometry. Phosphate groups are labile and easily lost during the processing of samples for mass spectrometry. Thus, effective methods to enrich for phosphopeptides from protein extracts have been developed. Here, we discuss and present in detail two such methods that we routinely employ. These methods are based on a sample enrichment step performed on titanium dioxide matrices followed by label-free tandem mass spectrometry and semi-quantitation.

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References

  1. Hardman G, Perkins S, Ruan Z, Kannan N, Brownridge P, Byrne DP, Eyers PA, Jones AR, Eyers CE (2017) Extensive non-canonical phosphorylation in human cells revealed using strong-anion exchange-mediated phosphoproteomics. bioRxiv

    Google Scholar 

  2. Cohen P (2002) The origins of protein phosphorylation. Nat Cell Biol 4(5):E127–E130. https://doi.org/10.1038/ncb0502-e127

    Article  CAS  PubMed  Google Scholar 

  3. Hunter T (2012) Why nature chose phosphate to modify proteins. Philos Trans R Soc Lond Ser B Biol Sci 367(1602):2513–2516. https://doi.org/10.1098/rstb.2012.0013

    Article  CAS  Google Scholar 

  4. Mashinchian O, Pisconti A, Le Moal E, Bentzinger CF (2018) The muscle stem cell niche in health and disease. Curr Top Dev Biol 126:23–65. https://doi.org/10.1016/bs.ctdb.2017.08.003

    Article  PubMed  Google Scholar 

  5. Olguin HC, Pisconti A (2012) Marking the tempo for myogenesis: Pax7 and the regulation of muscle stem cell fate decisions. J Cell Mol Med 16(5):1013–1025. https://doi.org/10.1111/j.1582-4934.2011.01348.x

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Pisconti A, Bernet JD, Olwin BB (2012) Syndecans in skeletal muscle development, regeneration and homeostasis. Muscles Ligaments Tendons J 2(1):1–9

    PubMed  PubMed Central  Google Scholar 

  7. Arecco N, Clarke CJ, Jones FK, Simpson DM, Mason D, Beynon RJ, Pisconti A (2016) Elastase levels and activity are increased in dystrophic muscle and impair myoblast cell survival, proliferation and differentiation. Sci Rep 6:24708. https://doi.org/10.1038/srep24708

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Ghadiali RS, Guimond SE, Turnbull JE, Pisconti A (2017) Dynamic changes in heparan sulfate during muscle differentiation and ageing regulate myoblast cell fate and FGF2 signalling. Matrix Biol 59:54–68. https://doi.org/10.1016/j.matbio.2016.07.007

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Segales J, Perdiguero E, Munoz-Canoves P (2016) Regulation of muscle stem cell functions: a focus on the p38 MAPK signaling pathway. Front Cell Dev Biol 4:91. https://doi.org/10.3389/fcell.2016.00091

    Article  PubMed  PubMed Central  Google Scholar 

  10. Huttlin EL, Jedrychowski MP, Elias JE, Goswami T, Rad R, Beausoleil SA, Villen J, Haas W, Sowa ME, Gygi SP (2010) A tissue-specific atlas of mouse protein phosphorylation and expression. Cell 143(7):1174–1189. https://doi.org/10.1016/j.cell.2010.12.001

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Zhou H, Di Palma S, Preisinger C, Peng M, Polat AN, Heck AJ, Mohammed S (2013) Toward a comprehensive characterization of a human cancer cell phosphoproteome. J Proteome Res 12(1):260–271. https://doi.org/10.1021/pr300630k

    Article  CAS  PubMed  Google Scholar 

  12. Johnson H, Eyers CE (2010) Analysis of post-translational modifications by LC-MS/MS. In: Cutillas PR, Timms JF (eds) LC-MS/MS in proteomics: methods and applications. Humana Press, Totowa, NJ, pp 93–108. https://doi.org/10.1007/978-1-60761-780-8_5

    Chapter  Google Scholar 

  13. Hogrebe A, von Stechow L, Bekker-Jensen DB, Weinert BT, Kelstrup CD, Olsen JV (2018) Benchmarking common quantification strategies for large-scale phosphoproteomics. Nat Commun 9(1):1045. https://doi.org/10.1038/s41467-018-03309-6

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Hawkridge AM (2014) Chapter 1 practical considerations and current limitations in quantitative mass spectrometry-based proteomics. In: Quantitative proteomics. The Royal Society of Chemistry, Cambridge, pp 1–25. https://doi.org/10.1039/9781782626985-00001

    Chapter  Google Scholar 

  15. Schmutz C, Ahrne E, Kasper CA, Tschon T, Sorg I, Dreier RF, Schmidt A, Arrieumerlou C (2013) Systems-level overview of host protein phosphorylation during Shigella flexneri infection revealed by phosphoproteomics. Mol Cell Proteomics 12(10):2952–2968. https://doi.org/10.1074/mcp.M113.029918

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Kauko O, Laajala TD, Jumppanen M, Hintsanen P, Suni V, Haapaniemi P, Corthals G, Aittokallio T, Westermarck J, Imanishi SY (2015) Label-free quantitative phosphoproteomics with novel pairwise abundance normalization reveals synergistic RAS and CIP2A signaling. Sci Rep 5:13099. https://doi.org/10.1038/srep13099

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Humphrey SJ, Yang G, Yang P, Fazakerley DJ, Stockli J, Yang JY, James DE (2013) Dynamic adipocyte phosphoproteome reveals that Akt directly regulates mTORC2. Cell Metab 17(6):1009–1020. https://doi.org/10.1016/j.cmet.2013.04.010

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Casado P, Alcolea MP, Iorio F, Rodriguez-Prados JC, Vanhaesebroeck B, Saez-Rodriguez J, Joel S, Cutillas PR (2013) Phosphoproteomics data classify hematological cancer cell lines according to tumor type and sensitivity to kinase inhibitors. Genome Biol 14(4):R37. https://doi.org/10.1186/gb-2013-14-4-r37

    Article  PubMed  PubMed Central  Google Scholar 

  19. Sano A, Nakamura H (2004) Titania as a chemo-affinity support for the column-switching HPLC analysis of phosphopeptides: application to the characterization of phosphorylation sites in proteins by combination with protease digestion and electrospray ionization mass spectrometry. Anal Sci 20(5):861–864

    Article  CAS  Google Scholar 

  20. Pinkse MW, Uitto PM, Hilhorst MJ, Ooms B, Heck AJ (2004) Selective isolation at the femtomole level of phosphopeptides from proteolytic digests using 2D-NanoLC-ESI-MS/MS and titanium oxide precolumns. Anal Chem 76(14):3935–3943. https://doi.org/10.1021/ac0498617

    Article  CAS  PubMed  Google Scholar 

  21. Thingholm TE, Jorgensen TJ, Jensen ON, Larsen MR (2006) Highly selective enrichment of phosphorylated peptides using titanium dioxide. Nat Protoc 1(4):1929–1935. https://doi.org/10.1038/nprot.2006.185

    Article  CAS  PubMed  Google Scholar 

  22. Sugiyama N, Masuda T, Shinoda K, Nakamura A, Tomita M, Ishihama Y (2007) Phosphopeptide enrichment by aliphatic hydroxy acid-modified metal oxide chromatography for nano-LC-MS/MS in proteomics applications. Mol Cell Proteomics 6(6):1103–1109. https://doi.org/10.1074/mcp.T600060-MCP200

    Article  CAS  PubMed  Google Scholar 

  23. Ferries S, Perkins S, Brownridge PJ, Campbell A, Eyers PA, Jones AR, Eyers CE (2017) Evaluation of parameters for confident phosphorylation site localization using an orbitrap fusion tribrid mass spectrometer. J Proteome Res 16(9):3448–3459. https://doi.org/10.1021/acs.jproteome.7b00337

    Article  CAS  PubMed  Google Scholar 

  24. Beausoleil SA, Villen J, Gerber SA, Rush J, Gygi SP (2006) A probability-based approach for high-throughput protein phosphorylation analysis and site localization. Nat Biotechnol 24(10):1285–1292. https://doi.org/10.1038/nbt1240

    Article  CAS  PubMed  Google Scholar 

  25. Paulo JA, McAllister FE, Everley RA, Beausoleil SA, Banks AS, Gygi SP (2015) Effects of MEK inhibitors GSK1120212 and PD0325901 in vivo using 10-plex quantitative proteomics and phosphoproteomics. Proteomics 15(2–3):462–473. https://doi.org/10.1002/pmic.201400154

    Article  CAS  PubMed  Google Scholar 

  26. Flamini V, Ghadiali RS, Antczak P, Rothwell A, Turnbull JE, Pisconti A (2018) The satellite cell niche regulates the balance between myoblast differentiation and self-renewal via p53. Stem Cell Rep 10(3):970–983. https://doi.org/10.1016/j.stemcr.2018.01.007

    Article  CAS  Google Scholar 

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Acknowledgments

This work was supported by a PhD studentship to FKJ, GEH and SF from the Biological and Biotechnology Research Council, UK and by a Wellcome Trust ISSF and a Marie Curie IEF to AP.

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Authors and Affiliations

  1. Department of Biochemistry, Institute of Integrative Biology, University of Liverpool, Liverpool, UK

    Fiona K. Jones, Gemma E. Hardman, Samantha Ferries, Claire E. Eyers & Addolorata Pisconti

  2. Centre for Proteome Research, Institute of Integrative Biology, University of Liverpool, Liverpool, UK

    Gemma E. Hardman, Samantha Ferries & Claire E. Eyers

Authors
  1. Fiona K. Jones
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  2. Gemma E. Hardman
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  3. Samantha Ferries
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  4. Claire E. Eyers
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  5. Addolorata Pisconti
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Corresponding author

Correspondence to Addolorata Pisconti .

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Editors and Affiliations

  1. Nofima AS, Ã…s, Norway

    Sissel Beate Rønning

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Jones, F.K., Hardman, G.E., Ferries, S., Eyers, C.E., Pisconti, A. (2019). Myoblast Phosphoproteomics as a Tool to Investigate Global Signaling Events During Myogenesis. In: Rønning, S. (eds) Myogenesis. Methods in Molecular Biology, vol 1889. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8897-6_18

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  • DOI: https://doi.org/10.1007/978-1-4939-8897-6_18

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-8896-9

  • Online ISBN: 978-1-4939-8897-6

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