Abstract
Glycoproteins have been a topic of interest for many years because of the important roles they play in many diverse biological functions such as molecular recognition. For many glycoproteins, the key to their functions are their oligosaccharide moieties. However, existing methods of analysis are often labor intensive or costly. Previously, my research group developed a method using high performance liquid chromatography with charged aerosol detection (HPLC-CAD) that was sensitive without requiring derivatization or exact standards. The earlier work used an amino column, but the method was limited in sensitivity due to column bleed and gave moderate resolution. In this presentation, I discuss the use of a porous graphitic carbon (PGC) column. A 3 µm particle diameter, 100 x 4 mm Hypercarb PGC column was used for these studies. Both reducing oligosaccharides and non-reducing oligosaccharides were investigated for use as calibration standards. The reducing oligosaccharides gave multiple peaks due to separation of alpha and beta anomers, making calibration difficult. Solutions of reduced oligosaccharide standards (linear chains and cyclodextrins) were made at different concentrations and analyzed by HPLC-CAD with the PGC column using gradient elution. The ion voltage of the home-built CAD instrument was optimized and calibration curves produced. Sugars reduced by sodium borohydride were able to test the methodology over a greater range of possible oligosaccharides and to match the reaction used to cleave oligosaccharides from glycoproteins. The reduction of oligosaccharides was a success, with near 100% reduction of maltotetraose to maltotetraitol using sodium borohydride and generally with good precision. The analysis of oligosaccharide standards on the PGC column showed excellent linearity with optimized ion voltages. The detection limit was found to be 0.2 ng, while previous results with the amino column had detection limits near 1 ng. The current method also resulted in two times the peak capacity due to higher separation efficiency. The PGC column has demonstrated improvements in the analysis of oligosaccharides by providing increased sensitivity and linearity over the amino column. Glycoproteins such as immunoglobulin G and ribonuclease B were used to test the method for analysis. Our method showed good chromatographic results. With further work, complete resolution of peaks may be obtained.