Phosphoprotein Enrichment for Protein Analysis in Motile T-Lymphocytes

  • Praseetha PrasannanEmail author
  • Rathi Saravanan
  • Navin Kumar Verma
Part of the Methods in Molecular Biology book series (MIMB, volume 1930)


Protein phosphorylation plays a key role in intracellular signal transduction and regulates diverse cellular functions. This posttranslational modification of proteins occurs dynamically and reversibly and only a small fraction of the total proteins is phosphorylated at any given time depending on the cell types and their functioning. Thus, a relatively low abundance of phosphorylated proteins is present in specific cells under certain conditions and hence it becomes problematic to detect these proteins and their analysis. In particular, phosphoproteomic analysis of rapidly migrating T-lymphocytes is always challenging. In order to analyze phosphoproteins in motile T-cells using techniques such as polyacrylamide gel electrophoresis and mass spectrometry, it is often important to enrich the phosphorylated forms in the cellular lysates. In this chapter, we describe a simple method to enrich phosphoproteins that can be used for protein analysis in motile T-cells.

Key words

Protein phosphorylation Phosphoproteomics T-cell migration Mass spectrometry 



This work was supported by the Lee Kong Chian School of Medicine, Nanyang Technological University Singapore Start-Up Grant to N.K.V. and the Singapore Ministry of Education (MOE) under its Singapore MOE Academic Research Fund (AcRF) Tier 2 Grant (MOE2017-T2-2-004).


  1. 1.
    Day EK, Sosale NG, Lazzara MJ (2016) Cell signaling regulation by protein phosphorylation: a multivariate, heterogeneous, and context-dependent process. Curr Opin Biotechnol 40:185–192CrossRefGoogle Scholar
  2. 2.
    Hunter T (1995) Protein kinases and phosphatases: the yin and yang of protein phosphorylation and signaling. Cell 80:225–236CrossRefGoogle Scholar
  3. 3.
    Ardito F, Giuliani M, Perrone D, Troiano G, Lo Muzio L (2017) The crucial role of protein phosphorylation in cell signaling and its use as targeted therapy. Int J Mol Med 40:271–280CrossRefGoogle Scholar
  4. 4.
    Van Hoof D, Muñoz J, Braam SR, Pinkse MW, Linding R, Heck AJ, Mummery CL, Krijgsveld J (2009) Phosphorylation dynamics during early differentiation of human embryonic stem cells. Cell Stem Cell 5:214–226CrossRefGoogle Scholar
  5. 5.
    Humphrey SJ, James DE, Mann M (2015) Protein phosphorylation: a major switch mechanism for metabolic regulation. Trends Endocrinol Metab 26:676–687CrossRefGoogle Scholar
  6. 6.
    Su B, Karin M (1996) Mitogen-activated protein kinase cascades and regulation of gene expression. Curr Opin Immunol 8:402–411CrossRefGoogle Scholar
  7. 7.
    Peterburs P, Heering J, Link G, Pfizenmaier K, Olayioye MA (2009) Hausser A (2009) Protein kinase D regulates cell migration by direct phosphorylation of the cofilin phosphatase slingshot 1 like. Cancer Res 69:5634–5638CrossRefGoogle Scholar
  8. 8.
    Serrano BP, Hardy JA (2018) Phosphorylation by protein kinase A disassembles the caspase-9 core. Cell Death Differ 25:1025–1039CrossRefGoogle Scholar
  9. 9.
    Kosako H, Nagano K (2011) Quantitative phosphoproteomics strategies for understanding protein kinase-mediated signal transduction pathways. Expert Rev Proteomics 8:81–94CrossRefGoogle Scholar
  10. 10.
    von Stechow L, Francavilla C, Olsen JV (2015) Recent findings and technological advances in phosphoproteomics for cells and tissues. Expert Rev Proteomics 12:469–487CrossRefGoogle Scholar
  11. 11.
    Choudhary C, Mann M (2010) Decoding signalling networks by mass spectrometry-based proteomics. Nat Rev Mol Cell Biol 11:427–439CrossRefGoogle Scholar
  12. 12.
    Guerin M, Gonçalves A, Toiron Y, Baudelet E, Audebert S, Boyer JB, Borg JP, Camoin L (2017) How may targeted proteomics complement genomic data in breast cancer? Expert Rev Proteomics 14:43–54CrossRefGoogle Scholar
  13. 13.
    Verma NK, Dempsey E, Freeley M, Botting CH, Long A, Kelleher D, Volkov Y (2011) Analysis of dynamic tyrosine phosphoproteome in LFA-1 triggered migrating T-cells. J Cell Physiol 226:1489–1498CrossRefGoogle Scholar
  14. 14.
    Ong ST, Freeley M, Skubis-Zegadło J, Fazil MH, Kelleher D, Fresser F, Baier G, Verma NK, Long A (2014) Phosphorylation of Rab5a protein by protein kinase Cϵ is crucial for T-cell migration. J Biol Chem 289:19420–19434CrossRefGoogle Scholar
  15. 15.
    Volkov Y, Long A, McGrath S, Ni Eidhin D, Kelleher D (2001) Crucial importance of PKC-beta(I) in LFA-1-mediated locomotion of activated T cells. Nat Immunol 2:508–514CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Praseetha Prasannan
    • 1
    Email author
  • Rathi Saravanan
    • 1
  • Navin Kumar Verma
    • 2
  1. 1.Lymphocyte Signalling Research Laboratory, Lee Kong Chian School of MedicineNanyang Technological University SingaporeSingaporeSingapore
  2. 2.Lee Kong Chian School of MedicineNanyang Technological University SingaporeSingaporeSingapore

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