A novel method for the separation and purification of human serum acid alpha-1-glycoprotein. Liquid chromatographic and mass spectrometric investigation of tryptic fragments

É Szöllosi, T. Kremmer, K. Ludányi, T. Imre, G. Schlosser, M. Boldizsár, B. Vincze, K. Vékey

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8 Citations (Scopus)

Abstract

Analytical ion-exchange chromatography resulted in significantly elevated human serum acid alpha-1-glycoprotein (AGP) levels in cancer patients (153-194 mgdL-1) and in acute inflammation (161 mgdL-1) compared to healthy individuals (58.5 ± 17.4 mgdL-1) and non-tumorous diseases (61.6 ± 5.0 mgdL-1). A novel method of sample preparation and dyeligand affinity chromatography was worked out and applied to the fast and biocompatible isolation and purification of AGP. Solvent extraction and precipitation provided nearly 100% recovery of serum AGP, while affinity chromatography using a new generation of dye-ligand phase (Toyopearl AF-Blue) resulted in AGP with a total yield over 85%. Tryptic fragments of AGP were separated by reversed phase liquid chromatography (RP-HPLC) and identified either with on-line ESI-MS, or off-line with PNGase F digestion and NH 2-phase HPLC. Off-line analysis provided further data on the distribution of antennary oligosaccharide structures. Predominance of bi- and triantennary chains was shown in human serum AGP (38 and 30%, respectively), while significant elevation in the relative proportion of tetraantennary compounds (9.6 vs 15.0%) and a 2-3 fold increase in the total amount of fucose containing structures were observed in cancer patients compared to the healthy controls.

Original languageEnglish
Pages (from-to)S213-S219
JournalChromatographia
Volume60
Issue numberSUPPL.
Publication statusPublished - Aug 20 2004

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Keywords

  • Column chromatography - mass spectrometry
  • Glycopeptides and oligosaccharides
  • Human serum acid alpha-1-glycoprotein (AGP)
  • Trypsin and endoglycosidase F digestion

ASJC Scopus subject areas

  • Analytical Chemistry
  • Biochemistry
  • Clinical Biochemistry
  • Organic Chemistry

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