Phosphoprotein enrichment from soluble and microsomal fractions of grapevine (Vitis vinifera cv. Gamay) cell culture using metal oxide affinity chromatography (MOAC)

Abstract:

Phosphorylation is one of the most prominent post-translational protein modifications in living cells and its investigation is of key interest in the field of proteomics. However, the frequently low stoichiometry of phosphorylation makes phosphoproteins harder to detect and identify. To overcome this problem, prefractionation methods of total cellular proteins are highly desirable. In this study, we have performed a phosphoenrichment at protein level by metal oxide affinity chromatography using Al (OH) 3 as a metal binding matrix for phosphate group capture [1]. Here, a strategy based on phosphoprotein enrichment by MOAC followed by a separation using a gel-based approach and phosphoprotein detection by specific fluorescence staining is shown. Phosphoprotein enrichment and subsequent protein separation using a gel-based approach (1D SDS-PAGE/2-DE) avoids the problem of complex protein mixtures analysed by gel-free technologies. Positive-stained proteins were identified by LC-MS/MS and phosphorylation sites were analysed using the automatic detection of neutral loss scan for H3PO4 [2].

Año de publicación:

2011

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