The analysis of phosphoproteomes by selective labelling and advanced mass spectrometric techniques.
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.
Amoresano
Angela
A
Department of Organic Chemistry and Biochemistry, Federico II University of Naples, Naples, Italy.
Cirulli
Claudia
C
Monti
Gianluca
G
Quemeneur
Eric
E
Marino
Gennaro
G
D000818:Animals; D006801:Humans; D007553:Isotope Labeling / Q000379:methods*; D013058:Mass Spectrometry / Q000379:methods* / Q000639:trends; D008954:Models, Biological; D010750:Phosphoproteins / Q000032:analysis*; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism; D020543:Proteome / Q000032:analysis*
D010750:Phosphoproteins; D020543:Proteome; D011494:Protein Kinases