Summary
This chapter focuses on the development of new proteomic approaches based on classical biochemical procedures coupled with new mass spectrometry methods to study the phosphorylation, the most important and abundant PTMs in modulating protein activity and propagating signals within cellular pathways and networks. These phosphoproteome studies aim at comprehensive analysis of protein phosphorylation by identification of the phosphoproteins, exact localization of phosphorylated residues, and preferably quantification of the phosphorylation. Because of low stoichiometry, heterogeneity, and low abundance, enrichment of phosphopeptides is an important step of this analysis. The first section is focused on the development of new enrichment methods coupled to mass spectrometry. Thus, improved approach, based on simple chemical manipulations and mass spectrometric procedures, for the selective analysis of phosphoserine and phosphothreonine in protein mixtures, following conversion of the peptide phosphate moiety into DTT derivatives, is described. However the major aim of this work is devoted to the use of isotopically labelled DTT, thus allowing a simple and direct quantitative MS analysis. The final part of the work is focused on the development of a strategy to study phosphorylation without preliminary enrichment but using the high performance of a novel hybrid mass spectrometer linear ion trap.
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Acknowledgments
Acknowledgments This work was supported by grants from the Ministero dell'Universita' e della Ricerca Scientifica (Progetti di Rilevante Interesse Nazionale 2002, 2003, 2005, 2006; FIRB 2001). Support from the National Center of Excellence in Molecular Medicine (MIUR – Rome) and from the Regional Center of Competence (CRdC ATIBB, Regione Campania – Naples) is gratefully acknowledged.
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Amoresano, A., Cirulli, C., Monti, G., Quemeneur, E., Marino, G. (2009). The Analysis of Phosphoproteomes by Selective Labelling and Advanced Mass Spectrometric Techniques. In: Graauw, M.d. (eds) Phospho-Proteomics. Methods in Molecular Biology™, vol 527. Humana Press. https://doi.org/10.1007/978-1-60327-834-8_13
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DOI: https://doi.org/10.1007/978-1-60327-834-8_13
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