Skip to main content

Phosphoproteomic Analysis of Soybean Roots Under Salinity by Using the iTRAQ Labeling Approach

  • Protocol
  • First Online:
Plant Phosphoproteomics

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2358))

  • 900 Accesses

Abstract

Protein phosphorylation is one of the most important posttranslational modifications. The phosphorylation and dephosphorylation of proteins regulate almost every cellular process, and the understanding of their functions can provide insights into the regulation of living systems at the molecular level. In recent years, both the rapid developments of enrichment approaches for phosphoproteins and MS techniques have improved the research scope and depth of phosphoproteomics. Using NaCl-treated soybean roots as the experimental materials, this chapter introduces the protein extraction, digestion with filter-aided sample preparation (FASP), eight-plex iTRAQ labeling, TiO2-based enrichment of phosphopeptides, LC-MS/MS analysis, as well as bioinformatic methods and protocols.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 109.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 139.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 219.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Havelund JF, Thelen JJ, Moller IM (2013) Biochemistry, proteomics, and phosphoproteomics of plant mitochondria from non-photosynthetic cells. Front Plant Sci 4:51. https://doi.org/10.3389/fpls.2013.00051

    Article  PubMed  PubMed Central  Google Scholar 

  2. Alonso A, Sasin J, Bottini N et al (2004) Protein tyrosine phosphatases in the human genome. Cell 117(6):699–711. https://doi.org/10.1016/j.cell.2004.05.018

    Article  CAS  PubMed  Google Scholar 

  3. Imanishi SY, Kochin V, Eriksson JE (2007) Optimization of phosphopeptide elution conditions in immobilized Fe(III) affinity chromatography. Proteomics 7(2):174–176. https://doi.org/10.1002/pmic.200600571

    Article  CAS  PubMed  Google Scholar 

  4. Nuhse TS, Stensballe A, Jensen ON et al (2003) Large-scale analysis of in vivo phosphorylated membrane proteins by immobilized metal ion affinity chromatography and mass spectrometry. Mol Cell Proteomics 2(11):1234–1243. https://doi.org/10.1074/mcp.T300006-MCP200

    Article  CAS  PubMed  Google Scholar 

  5. Conrads TP, Veenstra TD (2005) An enriched look at tyrosine phosphorylation. Nat Biotechnol 23(1):36–37. https://doi.org/10.1038/nbt0105-36

    Article  CAS  PubMed  Google Scholar 

  6. Maguire PB, Wynne KJ, Harney DF et al (2002) Identification of the phosphotyrosine proteome from thrombin activated platelets. Proteomics 2(6):642–648. https://doi.org/10.1002/1615-9861(200206)2:6<642::Aid-Prot642>3.0.Co;2-I

    Article  CAS  PubMed  Google Scholar 

  7. Steen H, Kuster B, Fernandez M et al (2002) Tyrosine phosphorylation mapping of the epidermal growth factor receptor signaling pathway. J Biol Chem 277(2):1031–1039. https://doi.org/10.1074/jbc.M109992200

    Article  CAS  PubMed  Google Scholar 

  8. Pi EX, Qu LQ, Hu JW et al (2016) Mechanisms of soybean Roots’ tolerances to salinity revealed by proteomic and phosphoproteomic comparisons between two cultivars. Mol Cell Proteomics 15(1):266–288. https://doi.org/10.1074/mcp.M115.051961

    Article  CAS  PubMed  Google Scholar 

  9. Pinkse MWH, Uitto PM, Hilhorst MJ et al (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 

  10. Beausoleil SA, Jedrychowski M, Schwartz D et al (2004) Large-scale characterization of HeLa cell nuclear phosphoproteins. Proc Natl Acad Sci U S A 101(33):12130–12135. https://doi.org/10.1073/pnas.0404720101

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Peck SC (2006) Phosphoproteomics in Arabidopsis: moving from empirical to predictive science. J Exp Bot 57(7):1523–1527. https://doi.org/10.1093/jxb/erj126

    Article  CAS  PubMed  Google Scholar 

  12. Sui SH, Wang JL, Cai Y et al (2007) Progress of technology and methodology in qauntitative phosphoproteomics. Prog Biochem Biophys 34(3):240–245

    CAS  Google Scholar 

  13. Zhen Y, Li CY, Lu Y et al (2014) Research progresses on phosphorylation of plant phosphoproteome. Genom Appl Biol 33(6):1405–1414

    Google Scholar 

  14. Pi EX, Xu J, Li HH et al (2019) Enhanced salt tolerance of rhizobia-inoculated soybean correlates with decreased phosphorylation of the transcription factor GmMYB183 and altered flavonoid biosynthesis. Mol Cell Proteomics 18(11):2225–2243. https://doi.org/10.1074/mcp.RA119.001704

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Pi EX, Zhu CM, Fan W et al (2018) Quantitative phosphoproteomic and metabolomic analyses reveal GmMYB173 optimizes flavonoid metabolism in soybean under salt stress. Mol Cell Proteomics 17(6):1209–1224. https://doi.org/10.1074/mcp.RA117.000417

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Vincent JM (1970) A manual for the practical study of root-nodule bacteria. IBP handbook no. 15. Published for the International Biological Programme by Blackwell Scientific, Oxford

    Google Scholar 

  17. Cox J, Mann M (2008) MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat Biotechnol 26(12):1367–1372. https://doi.org/10.1038/nbt.1511

    Article  CAS  PubMed  Google Scholar 

  18. Lv DW, Subburaj S, Cao M et al (2014) Proteome and phosphoproteome characterization reveals new response and defense mechanisms of Brachypodium distachyon leaves under salt stress. Mol Cell Proteomics 13(2):632–652. https://doi.org/10.1074/mcp.M113.030171

    Article  CAS  PubMed  Google Scholar 

  19. Nguyen TH, Brechenmacher L, Aldrich JT et al (2012) Quantitative phosphoproteomic analysis of soybean root hairs inoculated with Bradyrhizobium japonicum. Mol Cell Proteomics 11(11):1140–1155. https://doi.org/10.1074/mcp.M112.018028

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Ning DL, Liu KH, Liu CC et al (2016) Large-scale comparative phosphoprotein analysis of maize seedling leaves during greening. Planta 243(2):501–517. https://doi.org/10.1007/s00425-015-2420-3

    Article  CAS  PubMed  Google Scholar 

  21. Schwartz D, Gygi SP (2005) An iterative statistical approach to the identification of protein phosphorylation motifs from large-scale data sets. Nat Biotechnol 23(11):1391–1398. https://doi.org/10.1038/nbt1146

    Article  CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Erxu Pi .

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2021 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Qian, Y., Xu, J., Pi, E. (2021). Phosphoproteomic Analysis of Soybean Roots Under Salinity by Using the iTRAQ Labeling Approach. In: Wu, X.N. (eds) Plant Phosphoproteomics. Methods in Molecular Biology, vol 2358. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1625-3_5

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-1625-3_5

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-1624-6

  • Online ISBN: 978-1-0716-1625-3

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics