ovalbumin and 8-amino-1-3-6-naphthalenetrisulfonic-acid

Derivatization procedures using 1-phenyl-3-methyl-5-pyrazolone (PMP) and 2-aminonaphthalene trisulfone (ANTS) were selected among a number of well known methods for labelling carbohydrates. PMP derivatives were selected owing to our laboratory's previous high-performance liquid chromatography/electrospray ionization mass spectrometry (HPLC/ESI-MS) experience with these, whereas the ANTS-labelled compounds were prepared for fluorophore-assisted carbohydrate electrophoresis (FACE) separation. ANTS-oligosaccharide standards were characterized to study their ionization patterns. Reversed-phase and normal-phase HPLC systems were coupled on-line with ESI-MS. Each necessitated its own mobile phase system which, in turn, imposed some important changes in the ionization conditions used and/or on the ionization patterns and spectra obtained. Following characterization of the intact glycoprotein ovalbumin with ESI-MS, its glycans were detached using the enzyme PNGase-F. The glycans were subjected to PMP and ANTS derivatization. It was very difficult to separate ANTS derivatives by reversed-phase HPLC owing to lack of retention, and normal-phase HPLC offered reasonable retention with limited separation. PMP compounds overall yielded better normal- and reversed-phase separations and improved sensitivity over the ANTS-labelled sugars, for which negative mode ESI had to be used. The combination of ESI of intact ovalbumin and ESI of PMP-glycans gave rise to the detection of over 20 different glycoforms, excluding the possible presence of structural isomers for each sugar composition detected.

Topics: Amidohydrolases; Animals; Antipyrine; Carbohydrate Sequence; Chickens; Chromatography, High Pressure Liquid; Edaravone; Fluorescent Dyes; Molecular Sequence Data; Naphthalenes; Oligosaccharides; Ovalbumin; Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; Polysaccharides; Spectrometry, Mass, Electrospray Ionization

The effect of buffer conditions -- varying in salt type, pH, and concentration -- on the separation of 8-aminonaphthalene-1,3,6-trisulfonic acid (ANTS)-labeled monosaccharides and complex-type carbohydrates was investigated. Different buffer systems for high and low electroosmotic flow conditions were chosen: a phosphate and a citrate background electrolyte, each at pH 2.5, a phosphate buffer, pH 9.0, and a borate buffer at pH 9.5. All buffer systems displayed differences in resolution and selectivity. Phosphate and borate buffer demonstrated the greatest selectivity changes for ANTS-labeled carbohydrates. While separation in the phosphate system relies mainly on differences in the charge-to-mass-ratio, additional selectivity can be achieved with borate complexation of glycoconjugates. The use of borate buffers improved monosaccharide separations whereas complex carbohydrates showed a loss in resolution. The citrate background electrolyte at low pH caused no significant changes in the separation performance. The pH 9.0 phosphate buffer showed a reversed migration order of the ANTS conjugates with a decreased resolution, compared to the pH 2.5 phosphate buffer, due to the strong electroosmotic flow generated under high pH conditions. An ovalbumin-derived oligosaccharide library demonstrates the significance of buffer selectivity for complex carbohydrate separations. The separation in the acidic phosphate and the alkaline borate buffer generates a different pattern and only the combination of both buffer systems allows an appropriate assessment of sample complexity.

Topics: Acids; Alkalies; Animals; Borates; Buffers; Carbohydrate Conformation; Carbohydrate Sequence; Carbohydrates; Databases, Factual; Electrophoresis, Capillary; Molecular Sequence Data; Monosaccharides; Naphthalenes; Oligosaccharides; Ovalbumin; Phosphates

Complex oligosaccharides, both neutral and sialylated, were derivatized with 8-aminonaphthalene-1,3,6-trisulfonic acid (ANTS) and separated by capillary electrophoresis. The derivatization reaction was carried out in a total reaction volume of 2 microliters. The separated peaks were detected by laser-induced fluorescence detection using the 325-nm line of a He-Cd laser. Concentration and mass detection limits of 5 x 10(-8) M and 500 amol, respectively, could be achieved. The limiting step for higher sensitivity is not the detector performance, however, but the chemistry with a derivatization limit of 2.5 x 10(-6) M. Two labelling protocols were established, one with overnight reaction at 40 degrees C and the other with a 2.5-h derivatization time at 80 degrees C. Neutral oligosaccharides could be labelled with either protocol. However, sialylated oligosaccharides hydrolysed when labeled at 80 degrees C. Low nanomole to picomole amounts of oligomannose-type and complex-type oligosaccharide mixtures were derivatized and separated in less than 8 min with excellent resolution using a phosphate background electrolyte at pH 2.5. The linear relationship between the electrophoretic mobility and the charge-to-mass ratios of the ANTS conjugates was used for peak assignment. Further, the influence of the three-dimensional structure of the complex oligosaccharides on their migration behaviour is discussed. The suitability of the ANTS derivatization and the subsequent separation for the analysis of complex oligosaccharide patterns is demonstrated with oligosaccharide libraries derived from ovalbumin and bovine fetuin. For peak assignment the patterns are compared with those of the oligomannose and the complex-type oligosaccharide mixtures.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: alpha-Fetoproteins; Animals; Cattle; Coloring Agents; Electrophoresis, Capillary; Lasers; Naphthalenes; Oligosaccharides; Ovalbumin; Sialic Acids; Spectrometry, Fluorescence