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Two-Dimensional Gel Electrophoresis Approach for CTL Phosphoproteome Analysis

  • Maria Teresa RocchettiEmail author
  • Massimo Papale
  • Loreto Gesualdo
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1186)

Abstract

Phosphorylation of proteins plays a pivotal role in signal transduction processes, and it is a key regulator of many biological cell functions. Various strategies have been proposed for the study of phosphoproteome; most of them require a multi-step analysis and sophisticated equipment. Here we describe the two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) analysis of PBMC phosphoproteome using as preliminary enrichment step a simple phosphoprotein isolation by lanthanum chloride. This strategy can be most certainly applied to study the phosphoproteome of CTLs isolated from PBMCs. The phosphoproteome analysis of PBMCs, as well as of CTLs, may help to reveal the signaling pathways essential to their biological role in health and disease.

Key words

Phosphoproteome Two-dimensional gel electrophoresis Lanthanum chloride PBMC CTL 

Notes

Acknowledgment

The study was supported by the Grant of the Italian Ministry of Research and Education (FIRB2010-CAROMICS) to L.G. and E.R.

References

  1. 1.
    Fardilha M, Esteves SL, Korrodi-Gregorio L et al (2010) The physiological relevance of protein phosphatase 1 and its interacting proteins to health and disease. Curr Med Chem 17:3996–4017PubMedCrossRefGoogle Scholar
  2. 2.
    Reinders J, Sickmann A (2005) State-of-the-art in phosphoproteomics. Proteomics 5:4052–4061PubMedCrossRefGoogle Scholar
  3. 3.
    Wu S, Lourette NM, Tolic N et al (2009) An integrated top-down and bottom-up strategy for broadly characterizing protein isoforms and modifications. J Proteome Res 8:1347–1357PubMedCentralPubMedCrossRefGoogle Scholar
  4. 4.
    Schulemberg B, Goodman TN, Aggeler R et al (2004) Characterization of dynamic and steady-state protein phosphorylation using a fluorescent phosphoprotein gel stain and mass spectrometry. Electrophoresis 25:2526–2532CrossRefGoogle Scholar
  5. 5.
    Kaufmann H, Bailey JE, Fussenegge M (2001) Use of antibodies for detection of phosphorylated proteins separated by two-dimensional gel electrophoresis. Proteomics 1:194–199PubMedCrossRefGoogle Scholar
  6. 6.
    Jerry DH, Surendra D, David LT (2013) Informatics of protein and posttranslational modification detection via shotgun proteomics. Methods Mol Biol 1002:167–179CrossRefGoogle Scholar
  7. 7.
    McDonald WH, Yates JR 3rd (2002) Shotgun proteomics and biomarker discovery. Dis Markers 18:99–105PubMedCentralPubMedCrossRefGoogle Scholar
  8. 8.
    Cao L, Ding Y, Hung N et al (2012) Quantitative phosphoproteomics reveals SLP-76 dependent regulation of PAG and Src family kinases in T cells. PloS One 7:e46725PubMedCentralPubMedCrossRefGoogle Scholar
  9. 9.
    Iwai LK, Benoist C, Mathis D et al (2010) Quantitative phosphoproteomic analysis of T cell receptor signaling in diabetes prone resistant mice. J Proteome Res 9:3135–3145PubMedCentralPubMedCrossRefGoogle Scholar
  10. 10.
    Ruperez P, Gago-Martinez A, Burlingame AL et al (2012) Quantitative phosphoproteomic analysis reveals a role for serine and threonine kinases in the cytoskeletal reorganization in early T cell receptor activation in human primary T cells. Mol Cell Proteomics 11:171–186PubMedCentralPubMedCrossRefGoogle Scholar
  11. 11.
    Pink M, Verma N, Polato F et al (2011) Precipitation by lanthanum ions: a straightforward approach to isolating phosphoproteins. J Proteomics 75:375–383PubMedCrossRefGoogle Scholar
  12. 12.
    Mesko B, Poliska S, Nagy L (2011) Gene expression profiles in peripheral blood for the diagnosis of autoimmune diseases. Trends Mol Med 17:223–233PubMedCrossRefGoogle Scholar
  13. 13.
    Zhang Z, Li Y, Guan X, Yang X et al (2012) Human leukocyte antigen class I on peripheral blood mononuclear cells as a non-invasive biomarker for esophageal cancer. Dis Esophagus 25:273–278PubMedCrossRefGoogle Scholar
  14. 14.
    Pasinetti GM, Fivecoat H, Ho L (2010) Personalized medicine in traumatic brain injury. Psychiatr Clin North Am 33:905–913PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Maria Teresa Rocchetti
    • 2
    Email author
  • Massimo Papale
    • 1
  • Loreto Gesualdo
    • 2
  1. 1.Core Facility of Proteomics and Mass Spectrometry, Department of Surgery and Medical SciencesUniversity of FoggiaFoggiaItaly
  2. 2.Department of Emergency and Organ TransplantationUniversity of BariBariItaly

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