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Identification and Quantitation of Signal Molecule-Dependent Protein Phosphorylation

  • Arnoud Groen
  • Ludivine Thomas
  • Kathryn Lilley
  • Claudius Marondedze
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1016)

Abstract

Phosphoproteomics is a fast-growing field that aims at characterizing phosphorylated proteins in a cell or a tissue at a given time. Phosphorylation of proteins is an important regulatory mechanism in many cellular processes. Gel-free phosphoproteome technique involving enrichment of phosphopeptide coupled with mass spectrometry has proven to be invaluable to detect and characterize phosphorylated proteins. In this chapter, a gel-free quantitative approach involving 15N metabolic labelling in combination with phosphopeptide enrichment by titanium dioxide (TiO2) and their identification by MS is described. This workflow can be used to gain insights into the role of signalling molecules such as cyclic nucleotides on regulatory networks through the identification and quantification of responsive phospho(proteins).

Key words

Gel-free proteomics Mass spectrometry Phosphoproteomics Quantitation TiO2 enrichment 

References

  1. 1.
    Besant PG, Tan E, Attwood PV (2003) Mammalian protein histidine kinases. Int J Biochem Cell Biol 35:297–309PubMedCrossRefGoogle Scholar
  2. 2.
    Cohen P (2000) The regulation of protein function by multisite phosphorylation—a 25 year update. Trends Biochem Sci 25:596–601PubMedCrossRefGoogle Scholar
  3. 3.
    Dhanasekaran N, Reddy EP (1998) Signaling by dual specificity kinases. Oncogene 17:1447–1455PubMedCrossRefGoogle Scholar
  4. 4.
    Ubersax JA, Ferrell JE (2007) Mechanisms of specificity in protein phosphorylation. Nat Rev Mol Cell Biol 8:530–541PubMedCrossRefGoogle Scholar
  5. 5.
    Cieśla J, Frączyk T, Rode W (2011) Phosphorylation of basic amino acid residues in proteins: important but easily missed. Acta Biochim Pol 58:137–148PubMedGoogle Scholar
  6. 6.
    Nakashima K, Ito Y, Yamaguchi-Shinozaki K (2009) Transcriptional regulatory networks in response to abiotic stresses in Arabidopsis and grasses. Plant Physiol 149:88–95PubMedCrossRefGoogle Scholar
  7. 7.
    Heemskerk AAM, Busnel JM, Schoenmaker B, Derks RJE, Klychnikov O, Hensbergen PJ, Deelder AM, Mayboroda OA (2012) Ultra-low flow electrospray ionization-mass spectrometry for improved ionization efficiency in phosphoproteomics. Anal Chem 84:4552–4559PubMedCrossRefGoogle Scholar
  8. 8.
    Thelemann A, Petti F, Griffin G, Iwata K, Hunt T et al (2005) Phosphotyrosine signaling networks in epidermal growth factor receptor overexpressing squamous carcinoma cells. Mol Cell Proteomics 4:356–376PubMedCrossRefGoogle Scholar
  9. 9.
    Beausoleil SA, Jedrychowski M, Schwartz D, Elias JE, Villen J et al (2004) Large-scale characterization of HeLa cell nuclear phosphoproteins. Proc Natl Acad Sci U S A 101:12130–12135PubMedCrossRefGoogle Scholar
  10. 10.
    Larsen MR, Thingholm TE, Jensen ON, Roepstorff P, Jorgensen TJD (2005) Highly selective enrichment of phosphorylated peptides from peptide mixtures using titanium dioxide microcolumns. Mol Cell Proteomics 4:873–886PubMedCrossRefGoogle Scholar
  11. 11.
    Thingholm TE, Jorgensen TJ, Jensen ON, Larsen MR (2006) Highly selective enrichment of phosphorylated peptides using titanium dioxide. Nat Protoc 1:1929–1935PubMedCrossRefGoogle Scholar
  12. 12.
    Ficarro SB, McCleland ML, Stukenberg PT, Burke DJ, Ross MM et al (2002) Phosphoproteome analysis by mass spectrometry and its application to Saccharomyces cerevisiae. Nat Biotechnol 20:301–305PubMedCrossRefGoogle Scholar
  13. 13.
    Zheng HY, Hu P, Quinn DF, Wang YK (2005) Phosphotyrosine proteomic study of interferon alpha signaling pathway using a combination of immunoprecipitation and immobilized metal affinity chromatography. Mol Cell Proteomics 4:721–730PubMedCrossRefGoogle Scholar
  14. 14.
    Old WM, Meyer-Arendt K, Aveline-Wolf L, Pierce KG, Mendoza A et al (2005) Comparison of label-free methods for quantifying human proteins by shotgun proteomics. Mol Cell Proteomics 4:1487–1502PubMedCrossRefGoogle Scholar
  15. 15.
    Silva JC, Denny R, Dorschel C, Gorenstein MV, Li GZ et al (2006) Simultaneous qualitative and quantitative analysis of the Escherichia coli proteome: a sweet tale. Mol Cell Proteomics 5:589–607PubMedGoogle Scholar
  16. 16.
    Ross PL, Huang YN, Marchese JN, Williamson B, Parker K et al (2004) Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents. Mol Cell Proteomics 3:1154–1169PubMedCrossRefGoogle Scholar
  17. 17.
    Thompson A, Schafer J, Kuhn K, Kienle S, Schwarz J et al (2003) Tandem mass tags: a novel quantification strategy for comparative analysis of complex protein mixtures by MS/MS. Anal Chem 75:1895–1904PubMedCrossRefGoogle Scholar
  18. 18.
    Conrads TP, Alving K, Veenstra TD, Belov ME, Anderson GA et al (2001) Quantitative analysis of bacterial and mammalian proteomes using a combination of cysteine affinity tags and 15N-metabolic labeling. Anal Chem 73:2132–2139PubMedCrossRefGoogle Scholar
  19. 19.
    Krijgsveld J, Ketting RF, Mahmoudi T, Johansen J, Artal-Sanz M et al (2003) Metabolic labeling of C. elegans and D. melanogaster for quantitative proteomics. Nat Biotechnol 21:927–931PubMedCrossRefGoogle Scholar
  20. 20.
    Ong SE, Blagoev B, Kratchmarova I, Kristensen DB, Steen H et al (2002) Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics. Mol Cell Proteomics 1:376–386PubMedCrossRefGoogle Scholar
  21. 21.
    Haegler K, Mueller NS, Maccarrone G, Hunyadi-Gulyas E, Webhofer C et al (2009) QuantiSpec—quantitative mass spectrometry data analysis of N-15-metabolically labeled proteins. J Proteomics 71:601–608PubMedCrossRefGoogle Scholar
  22. 22.
    Pan CL, Kora G, McDonald WH, Tabb DL, VerBerkmoes NC et al (2006) ProRata: a quantitative proteomics program for accurate protein abundance ratio estimation with confidence interval evaluation. Anal Chem 78:7121–7131PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  • Arnoud Groen
    • 1
  • Ludivine Thomas
    • 2
  • Kathryn Lilley
    • 1
  • Claudius Marondedze
    • 1
  1. 1.Department of Biochemistry, Cambridge Centre for Proteomics, Cambridge Systems Biology CentreUniversity of CambridgeCambridgeUK
  2. 2.Division of Chemical and Life Sciences and EngineeringKing Abdullah University of Science and TechnologyThuwalSaudi Arabia

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