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• W2001776916 abstract "The study of protein phosphorylation has grown exponentially in recent years, as it became evident that important cellular functions are regulated by phosphorylation and dephosphorylation of proteins on serine, threonine and tyrosine residues. The use of immobilized metal affinity chromatography (IMAC) to enrich phosphopeptides from peptide mixtures has been shown to be useful especially prior to mass spectrometric analysis. For the selective enrichment applying solid-phase extraction (SPE) of phosphorylated peptides, we introduce poly(glycidyl methacrylate/divinylbenzene) (GMD) derivatized with imino-diacetic acid (IDA) and bound FeIII as a material. GMD is rapidly synthesized and the resulting free epoxy groups enable an easy access to further derivatization with, e.g., IDA. Electron microscopy showed that the synthesized GMD-IDA-FeIII for SPE has irregular agglomerates of spherical particles. Inductively coupled plasma (ICP) analysis resulted in a metal capacity of FeIII being 25.4 μmol/mL. To enable on-line preconcentration and desalting in one single step, GMD-IDA-FeIII and Silica C18 were united in one cartridge. Methyl esterification (ME) of free carboxyl groups was carried out to prevent binding of nonphosphorylated peptides to the IMAC function. The recovery for a standard phosphopeptide using this SPE method was determined to be 92%. The suitability of the established system for the selective enrichment and analysis of model proteins phosphorylated at different amino acid residues was evaluated stepwise. After successful enrichment of β-casein deriving phosphopeptides, the established system was extented to the analysis of in vitro phosphorylated proteins, e.g. deriving from glutathione-S-transferase tagged extracellular signal regulated kinase 2 (GST-ERK2). Keywords: glycidyl methacrylate/divinylbenze • immobilized metal affinity chromatography • phosphopeptides • solid-phase extraction • mass spectrometry • GST-ERK2" @default.
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• W2001776916 date "2005-10-21" @default.
• W2001776916 modified "2023-10-02" @default.
• W2001776916 title "Poly(Glycidyl Methacrylate/Divinylbenzene)-IDA-Fe^III in Phosphoproteomics" @default.
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• W2001776916 doi "https://doi.org/10.1021/pr050224m" @default.
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