Highly sensitive derivatization reagents possessing positively charged structures for the determination of oligosaccharides in glycoproteins by high performance liquid chromatography electrospray ionization tandem mass spectrometry.
We have developed three kinds of novel derivatization reagents (4 CEBTPP, 4 CBBTPP, 5 COTPP) with triphenylphosphine (TPP) as a basic structure carrying a permanent positive charge for resolution of the oligosaccharides in glycoprotein using high performance liquid chromatography electrospray ionization tandem mass spectrometry (LC ESI MS/MS) The synthesized reagents reacted with the sialylglycosylamine of the sialylglycopeptide after treatment by PNGase F. The final derivatives were analyzed by ESI MS and sensitively detected in the selected reaction monitoring (SRM) mode. Furthermore, the limits of detection (S/N=3) on the SRM chromatograms were at the fmol level (30fmol) Therefore, we used the limit of detection of the reagent products detected by the SRM and evaluated the utility of each reagent. Among the reagents, the positively charged 4 CEBTPP derivative's peak area was the highest; 4 CEBTPP with a positively charged structure showed about a 20 times greater sensitivity for the glycosylamine of the SGP product compared to the conventional fluorescence reagent, Fmoc Cl. In addition, various fragment ions based on the carbohydrate units also appeared in the MS/MS spectra. Among the fragment ions, m/z 627.37 (CE=40eV) corresponding to 4 CEBTPP GlcNAc and m/z 120.09 (CE=100eV) corresponding to 4 CEBTPP are the most important ones for identifying the oligosaccharide. 4 CEBTPP SGA was easily identified by the selected ion chromatogram in the product ion scan (m/z 120.09) and in the precursor ion scan (m/z 627.37) by MS/MS detection. The derivatized analytes have a high ionization efficiency and they are detected with a high sensitivity in the electrospray ionization. The novel derivatization reagent with a multi function provided a higher sensitivity for the oligosaccharide analysis, as well as a better specificity and feasibility. Furthermore, several oligosaccharides in fetuin and ribonuclease B were successfully identified by the proposed procedure.
Journal of chromatography. A