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 bacterium Francisella 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 of Francisella tularensis glycoproteins. Effectiveness of the newly prepared magnetic microspheres for glycoprotein enrichment was proved by comparison with commercial hydrazide-functionalized microparticles.

Department of Chemistry Indiana University 800 E Kirkwood Avenue 47405 Bloomington IN USA

Institute of Macromolecular Chemistry Academy of Sciences of the Czech Republic Heyrovsky Sq 2 162 06 Prague Czech Republic

Institute of Molecular Pathology Faculty of Military Health Sciences University of Defence T řebešská 1575 500 01 Hradec Králové Czech Republic

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