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Title
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Development of a novel chemical probe for the selective enrichment of phosphorylated serine- and threonine-containing peptides
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Author
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Abstract
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| Gaining insight into phosphoproteomes is of the utmost importance for understanding regulation processes such as signal transduction and differentiation. While the identification of phosphotyrosine-containing amino acid sequences in peptides and proteeins is now becoming possible, mainly because of the availability of high affinity antibodies, no general and robust methodology allowing selelective enrichment and analysis of serine- and threonine-phoshorylated proteins and peptides is presently available. the method presented here involves chemical modification of phosphoylated serine or threonin residues and their subsequent derivatization with the aid of a multifunctional probe molecule. The designed probe contains four parts: a reactive group that is used to bind specifically to the modified phosphopeptide, an optional part in which heavy isotopes can be incorporated, an acid-labile tinker, and an affinity tog for the selective enrichment of modified'phosphopeptides from complex mixtures. The acid-cleavable linker allows full recovery from the offinity-purified material and removal of the affinity tag prior to MS analysis. The preparation of a representative probe molecule containing a biotin affinity tag and its applicability in phosphoproteome analysis is shown in a number of well-defined model systems of increasing degrees of complexity. Amounts of phosphopeptide as low as 1 nmol con be modified and enriched from a mixture of peptides. During the development of the beta-elimination/nucleophilic addition protocol, special attention was paid to the different experimental parameters that might affect the chemical-modification steps carried out on phosphoryloted residues. |
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Language
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English
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Source (journal)
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ChemBioChem: a European journal of chemical biology. - Weinheim
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Publication
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Weinheim
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Wiley
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2005
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ISSN
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1439-4227
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DOI
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10.1002/CBIC.200500209
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Volume/pages
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6
:12
(2005)
, p. 2271-2280
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ISI
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000233916600023
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Full text (Publisher's DOI)
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