Mapping phosphorylation sites: a new strategy based on the use of isotopically labelled DTT and mass spectrometry

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Eur. J. Mass Spectrom. DOI: 10.1255/ejms.599

Mapping phosphorylation sites: a new strategy based on the use of isotopically labelled DTT and mass spectrometry

Angela Amoresano and Gennaro Marino
Department of Organic Chemistry and Biochemistry, Federico II University of Naples, Naples, Italy and School of Biotechnological Sciences, Federico II University of Naples, Naples, Italy
Claudia Cirulli
Department of Organic Chemistry and Biochemistry, Federico II University of Naples, Naples, Italy
Eric Quemeneur
Department of Protein Engineering, Life Science Division, C.E.A., Marcoule, France

ABSTRACT:

Phosphoproteomics nowadays represent a front line in functional proteomics as testify by the number of papers recently appeared 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 from the phosphate esters the corresponding alkene moiety. This was achieved a) by separating the elimination reaction from the addition; b) by the use of Ba(OH)₂ as alkali reagent; and c) its further elimination by the mere addition of solid CO₂ to the peptide mixture. The Michael reaction was then performed, after the removal of BaCO₃ by centrifugation, by adding to the peptide mixture dithiothreitol (DTT). Finally the direct purification of the modified phosphopeptides was performed on a thiol-sepharose column. Furthermore the availability of fully deuterated DTT, thus introducing a 6 Da difference with respect to the light species, allows to quantitate the differential extent of signalling modification when analysed by MALDIMS and LC/MS. The entire procedure has been set up by using bovine alpha casein, and resulted in the identification of all the phosphorylated triptic peptides, including the tetraphosphorylated one, which escaped to all previous reported procedures. In conclusion this paper reports an integrated simple methodology which involves chemical replacement of the phosphate moieties by affinity tags. This method to locate and quantitate phosphorylated residues. Should be of widespread utility for defining signalling pathways and control mechanisms that involve phosphorylation or dephosphorylation of serine/threonine residues.

Keywords: alpha casein, phosphoproteome, ICAT, mass spectrometry

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