title: Mapping phosphorylation sites: a new strategy based on the use of isotopically labelled DTT and mass spectrometry.
abstract: Phosphoproteomics, nowadays, represents a front line in functional proteomics as testified by the number of papers recently appearing in the literature. In an attempt to improve and simplify the methods so far suggested we have set up a simple isotope-coded approach to label and quantitate phospho-Ser/-Thr residues in protein mixtures. First of all, after appropriate oxidation of cysteine/cystine residues followed by tryptic hydrolysis, we have optimised and simplified the beta-elimination reaction to get the corresponding alkene moiety from the phosphate esters. This was achieved by (a) separating the elimination reaction from the addition reaction, (b) the use of Ba(OH)(2) as alkali reagent and (c) its further elimination by the simple addition of solid CO(2) to the peptide mixture. The Michael reaction was then performed, after the removal of BaCO(3) by centrifugation, by adding dithiothreitol (DTT) to the peptide mixture. Finally, the direct purification of the modified phosphopeptides was performed on a thiol-sepharose column. The availability of fully deuterated DTT, introducing a 6 Da difference with respect to the non-deuterated species, allows quantitation of the differential extent of signalling modification when analysed by matrix-assisted laser desorption/ionisation mass spectrometry (MALDI-MS) and liquid chromatography/mass spectrometry. The entire procedure has been set up by using bovine alpha-casein, and resulted in the identification of all the phosphorylated tryptic peptides, including the tetraphosphorylated peptides, which escaped all previously reported procedures
authors.affiliation: Department of Organic Chemistry and Biochemistry, and School of Biotechnological Sciences, Federico II University of Naples, Naples, Italy.
authors.forename: Angela
authors.initials: A
authors.lastname: Amoresano
authors.forename: Gennaro
authors.initials: G
authors.lastname: Marino
authors.forename: Claudia
authors.initials: C
authors.lastname: Cirulli
authors.forename: Eric
authors.initials: E
authors.lastname: Quemeneur
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002364:Caseins / Q000737:chemistry / Q000378:metabolism; D002417:Cattle; D004229:Dithiothreitol / Q000737:chemistry*; D007553:Isotope Labeling; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010750:Phosphoproteins / Q000737:chemistry* / Q000378:metabolism; D010766:Phosphorylation; D040901:Proteomics / Q000379:methods; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013438:Sulfhydryl Compounds / Q000737:chemistry; D014357:Trypsin / Q000378:metabolism
chemical_list: D002364:Caseins; D010446:Peptide Fragments; D010750:Phosphoproteins; D013438:Sulfhydryl Compounds; D014357:Trypsin; D004229:Dithiothreitol