Reversible phosphorylation plays a key role in numerous biological processes. Mass spectrometry-based approaches are commonly used to analyze protein phosphorylation, but such analysis is challenging, largely due to the low phosphorylation stoichiometry. Hence, a number of phosphopeptide enrichment strategies have been developed, including metal oxide affinity chromatography (MOAC). Here, we describe a new material for performing MOAC that employs a magnetite-doped polydimethylsiloxane (PDMS), that is suitable for the creation of microwell array and microfluidic systems to enable low volume, high throughput analysis. Incubation time and sample loading were explored and optimized and demonstrate that the embedded magnetite is able to enrich phosphopeptides. This substrate-based approach is rapid, straightforward and suitable for simultaneously performing multiple, low volume enrichments.
Bibliographical noteFunding: RASOR (“Radical Solutions for Researching the proteome”) BBSRC IRC programme (BB/C511572/1) and the Proxomics EPSRC Cross-Disciplinary Research Landscape Award (EP/I017887/1).
Electronic supplementary information: DOI 10.1039/c4an00750f