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).
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Besant PG, Tan E, Attwood PV (2003) Mammalian protein histidine kinases. Int J Biochem Cell Biol 35:297–309
Cohen P (2000) The regulation of protein function by multisite phosphorylation—a 25 year update. Trends Biochem Sci 25:596–601
Dhanasekaran N, Reddy EP (1998) Signaling by dual specificity kinases. Oncogene 17:1447–1455
Ubersax JA, Ferrell JE (2007) Mechanisms of specificity in protein phosphorylation. Nat Rev Mol Cell Biol 8:530–541
Cieśla J, Frączyk T, Rode W (2011) Phosphorylation of basic amino acid residues in proteins: important but easily missed. Acta Biochim Pol 58:137–148
Nakashima K, Ito Y, Yamaguchi-Shinozaki K (2009) Transcriptional regulatory networks in response to abiotic stresses in Arabidopsis and grasses. Plant Physiol 149:88–95
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 84:4552–4559
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 4:356–376
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 101:12130–12135
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 4:873–886
Thingholm TE, Jorgensen TJ, Jensen ON, Larsen MR (2006) Highly selective enrichment of phosphorylated peptides using titanium dioxide. Nat Protoc 1:1929–1935
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 20:301–305
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 4:721–730
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 4:1487–1502
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 5:589–607
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 3:1154–1169
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 75:1895–1904
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 73:2132–2139
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 21:927–931
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 1:376–386
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 71:601–608
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 78:7121–7131
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2013 Springer Science+Business Media New York
About this protocol
Cite this protocol
Groen, A., Thomas, L., Lilley, K., Marondedze, C. (2013). Identification and Quantitation of Signal Molecule-Dependent Protein Phosphorylation. In: Gehring, C. (eds) Cyclic Nucleotide Signaling in Plants. Methods in Molecular Biology, vol 1016. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-441-8_9
Download citation
DOI: https://doi.org/10.1007/978-1-62703-441-8_9
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-62703-440-1
Online ISBN: 978-1-62703-441-8
eBook Packages: Springer Protocols