Development of a novel chemical probe for the selective enrichment of phosphorylated serine- and threonine-containing peptides

van der Veken, P., Dirksen, E.H.C., Ruijter, E., Elgersma, R.C., Heck, A.J.R., Rijkers, D.T.S., Slijper, M. and Liskamp, R.M.J. (2005) Development of a novel chemical probe for the selective enrichment of phosphorylated serine- and threonine-containing peptides. ChemBioChem, 6(12), pp. 2271-2280. (doi: 10.1002/cbic.200500209)

Abstract

Gaining insight into phosphoproteomes is of the utmost importance for understanding regulation processes such as signal transduction and cellular differentiation. While the identification of phosphotyrosine-containing amino acid sequences in peptides and proteins is now becoming possible, mainly because of the availability of high-affinity antibodies, no general and robust methodology allowing the selective enrichment and analysis of serine- and threonine-phosphorylated proteins and peptides is presently available. The method presented here involves chemical modification of phosphorylated serine or threonine 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 linker, and an affinity tag for the selective enrichment of modified phosphopeptides from complex mixtures. The acid-cleavable linker allows full recovery from the affinity-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 can be modified and enriched from a mixture of peptides. During the development of the β-elimination/nucleophilic addition protocol, special attention was paid to the different experimental parameters that might affect the chemical-modification steps carried out on phosphorylated residues.

• Item Type: Articles
• Status: Published
• Refereed: Yes
• Glasgow Author(s) Enlighten ID: Liskamp, Professor Robert
• Authors: van der Veken, P., Dirksen, E.H.C., Ruijter, E., Elgersma, R.C., Heck, A.J.R., Rijkers, D.T.S., Slijper, M., and Liskamp, R.M.J.
• College/School: College of Science and Engineering > School of Chemistry
• Journal Name: ChemBioChem
• ISSN: 1439-4227
• ISSN (Online): 1439-7633