Poly(glycidyl methacrylate/divinylbenzene)-IDA-FeIII in phosphoproteomics

被引:65
作者
Aprilita, NH
Huck, CW
Bakry, R
Feuerstein, I
Stecher, G
Morandell, S
Huang, HL
Stasyk, T
Huber, LA
Bonn, GK
机构
[1] Leopold Franzens Univ, Inst Analyt Chem & Radiochem, A-6020 Innsbruck, Austria
[2] Innsbruck Med Univ, Bioctr, Div Cell Biol, A-6020 Innsbruck, Austria
[3] Gadjah Mada Univ, Fac Math & Nat Sci, Dept Chem, Analyt Chem Lab, Yogyakarta, Indonesia
关键词
glycidyl methacrylate/divinylbenze; immobilized metal affinity chromatography; phosphopeptides; solid-phase extraction; mass spectrometry; GST-ERK2;
D O I
10.1021/pr050224m
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
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 Fe-III 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-Fe-III for SPE has irregular agglomerates of spherical particles. Inductively coupled plasma (ICP) analysis resulted in a metal capacity of Fe-III being 25.4 mu mol/mL. To enable on-line preconcentration and desalting in one single step, GMD-IDA-Fe-III 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 beta-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).
引用
收藏
页码:2312 / 2319
页数:8
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