Sugar Nucleotide Quantification Using Multiple Reaction Monitoring (MRM) LC-MSMS

Sugar nucleotides are monosaccharide derivatives involved in the glycosylation of proteins and lipids. The glycosylation process of proteins and lipids involves several steps such as the importing of nutrient monosaccharides to the cells prior to their conversion to sugar nucleotides. These structures are ultimately used in ER/Golgi as the "building block" of any glycan structures attached to proteins or lipids.  Aberrant glycosylation has been implicated with many diseases, yet it is not clear which of the glycosylation steps is(are) responsible for the aberrant glycosylation observed in disease states if not all. We have developed an RSLC tandem mass spectrometry method that allows for rapid separation while utilizing a multiple reaction monitoring approach to reliably quantify sugar nucleotides. Reliable Quantification of sugar nucleotides is achieved using MRM mode of LC-MSMS. Seven sugar nucleotides commonly associated with protein glycosylation (UDP-Gal, UDP-Glc, CMP-NeuAc, UDP-GalNac, UDP-GlcNac, GDP-Man, and GDP-Fuc) were separated utilizing a Hypercarb PGC column (2.1 mm x 100 mm). Separation was attained using the Ultimate 3000 rapid separation liquid chromatography instrument (RSLC). The effluent of this instrument was interfaced to a Vantage TSQ triple-quadrupole mass spectrometer. The method allowed the detection of sugar nucleotides at 100-200 pg on column with a linear dynamic range expanding over 5 orders of magnitude. Sugar nucleotides derived from different cancer cell lines with different response to drug treatment will be quantified in the future using this technique. With this method we hope to help in the elucidation of the onset of cancer.