title: Use of magnetic hydrazide-modified polymer microspheres for enrichment ofFrancisella tularensisglycoproteins.
abstract: The field of microbial proteomics has currently experienced a boom in the discovery of glycosylated proteins of various pathogenic bacteria as potential mediators of host-pathogen interactions. The presence of glycoproteins has recently been discovered in a Gram-negative pathogenic bacteriumFrancisella tularensis, utilizing glycoprotein detection and isolation techniques in combination with mass spectrometry. The isolation of glycoproteins is a prerequisite for their subsequent mass-spectrometric identification. Current glycoprotein isolation/enrichment methods comprise lectin affinity chromatography, aminophenylboronic acid and hydrazide-based enrichment. The use of magnetic microspheres containing functional groups is nowadays among state-of-art separation methodologies owing to an ease of manipulation, a speed of separation, and a minimum of non-specific protein adsorption. In the present study, novel magnetic hydrazide-modified poly(2-hydroxyethyl methacrylate) (PHEMA) microspheres were developed using a multi-step swelling and polymerization method with subsequent precipitation of magnetic iron oxides within the pores of the particles. The microspheres had a regular shape, size of 4 μm and contained 0.18 mmol hydrazide groups per g; the magnetic microspheres were employed for specific enrichment ofFrancisella tularensisglycoproteins. Effectiveness of the newly prepared magnetic microspheres for glycoprotein enrichment was proved by comparison with commercial hydrazide-functionalized microparticles.
authors.affiliation: Institute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic, Heyrovsky Sq. 2, 162 06 Prague, Czech Republic.
authors.forename: Daniel
authors.initials: D
authors.lastname: Horák
authors.affiliation: Institute of Molecular Pathology, Faculty of Military Health Sciences, University of Defence, T řebešská 1575, 500 01 Hradec Králové, Czech Republic.
authors.forename: Lucie
authors.initials: L
authors.lastname: Balonová
authors.affiliation: Department of Chemistry, Indiana University, 800 E Kirkwood Avenue, 47405 Bloomington, IN, USA.
authors.forename: Benjamin F
authors.initials: BF
authors.lastname: Mann
authors.affiliation: Institute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic, Heyrovsky Sq. 2, 162 06 Prague, Czech Republic.
authors.forename: Zdeněk
authors.initials: Z
authors.lastname: Plichta
authors.affiliation: Institute of Molecular Pathology, Faculty of Military Health Sciences, University of Defence, T řebešská 1575, 500 01 Hradec Králové, Czech Republic.
authors.forename: Lenka
authors.initials: L
authors.lastname: Hernychová
authors.affiliation: Department of Chemistry, Indiana University, 800 E Kirkwood Avenue, 47405 Bloomington, IN, USA.
authors.forename: Milos V
authors.initials: MV
authors.lastname: Novotny
authors.affiliation: Institute of Molecular Pathology, Faculty of Military Health Sciences, University of Defence, T řebešská 1575, 500 01 Hradec Králové, Czech Republic.
authors.forename: Jiří
authors.initials: J
authors.lastname: Stulík