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Issue 6, 2011
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Enrichment and sequencing of phosphopeptides on indium tin oxide coated glass slides

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Abstract

Unambiguous identification of phosphorylation sites is of premier importance to biologists, who seek to understand the role of phosphorylation from the perspective of site-specific control of biological phenomena. Despite this widely asked and highly specific information, many methods developed are aimed at analysis of complete proteomes, indeed even phospho-proteomes, surpassing the basic requests of many biologists. We have therefore further developed a simple method that specifically deals with the analysis of multiple phosphorylation sites on singular proteins or small collections of proteins. With this method, the whole purification process, from sample application to MALDI-MS analysis, can be performed on commercially available indium tin oxide (ITO) coated glass slides. We show that fifteen (15) samples can be purified within one hour, and that low femtomole sensitivity can be achieved. This limit of identification is demonstrated by the successful MS/MS-based identification of 6 fmol of monophosphopeptide from β-casein. We demonstrate that the method can be applied for identifying phosphorylation sites from recombinant and cell-derived biological protein samples. Since ITO-coated glass slides are inexpensive and available from several suppliers the method is readily and inexpensively available to other researchers. Taken together, the presented protocols and materials render this method as an extremely fast and sensitive phosphopeptide identification protocol that should aid biologists in discovery and validation of phosphorylation sites.

Graphical abstract: Enrichment and sequencing of phosphopeptides on indium tin oxide coated glass slides

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Publication details

The article was received on 08 Nov 2010, accepted on 30 Mar 2011 and first published on 27 Apr 2011


Article type: Method
DOI: 10.1039/C0MB00269K
Mol. BioSyst., 2011,7, 1828-1837

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    Enrichment and sequencing of phosphopeptides on indium tin oxide coated glass slides

    P. Kouvonen, E. Rainio, V. Suni, P. Koskinen and G. L. Corthals, Mol. BioSyst., 2011, 7, 1828
    DOI: 10.1039/C0MB00269K

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