title: Poly(glycidyl methacrylate/divinylbenzene)-IDA-FeIII in phosphoproteomics.
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 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 micromol/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 extended to the analysis of in vitro phosphorylated proteins, e.g. deriving from glutathione-S-transferase tagged extracellular signal regulated kinase 2 (GST-ERK2).
authors.affiliation: Institute of Analytical Chemistry and Radiochemistry, Leopold-Franzens University, Innsbruck, Austria.
authors.forename: Nurul H
authors.initials: NH
authors.lastname: Aprilita
authors.forename: Christian W
authors.initials: CW
authors.lastname: Huck
authors.forename: Rania
authors.initials: R
authors.lastname: Bakry
authors.forename: Isabel
authors.initials: I
authors.lastname: Feuerstein
authors.forename: Guenther
authors.initials: G
authors.lastname: Stecher
authors.forename: Sandra
authors.initials: S
authors.lastname: Morandell
authors.forename: Hong-Lei
authors.initials: HL
authors.lastname: Huang
authors.forename: Taras
authors.initials: T
authors.lastname: Stasyk
authors.forename: Lukas A
authors.initials: LA
authors.lastname: Huber
authors.forename: Guenther K
authors.initials: GK
authors.lastname: Bonn
mesh_terms: D000595:Amino Acid Sequence; D002364:Caseins / Q000737:chemistry; D002846:Chromatography, Affinity; D004852:Epoxy Compounds / Q000737:chemistry; D005982:Glutathione Transferase / Q000378:metabolism; D007098:Imino Acids / Q000737:chemistry; D007501:Iron / Q000032:analysis* / Q000737:chemistry; D013058:Mass Spectrometry; D008689:Methacrylates / Q000737:chemistry; D008854:Microscopy, Electron; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D008956:Models, Chemical; D008969:Molecular Sequence Data; D009211:Myoglobin / Q000737:chemistry; D010455:Peptides / Q000737:chemistry; D010748:Phosphopeptides / Q000737:chemistry; D010750:Phosphoproteins / Q000737:chemistry*; D010766:Phosphorylation; D011108:Polymers / Q000737:chemistry; D020543:Proteome; D040901:Proteomics / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013997:Time Factors; D014357:Trypsin / Q000494:pharmacology; D014753:Vinyl Compounds / Q000737:chemistry
chemical_list: D002364:Caseins; D004852:Epoxy Compounds; D007098:Imino Acids; D008689:Methacrylates; D009211:Myoglobin; D010455:Peptides; D010748:Phosphopeptides; D010750:Phosphoproteins; D011108:Polymers; D020543:Proteome; D014753:Vinyl Compounds; D007501:Iron; D005982:Glutathione Transferase; D019950:Mitogen-Activated Protein Kinase 1; D014357:Trypsin; C004985:divinyl benzene; C007870:glycidyl methacrylate; C008109:iminodiacetic acid