Quantification of monosaccharides through multiple-reaction monitoring liquid chromatography/mass spectrometry using an aminopropyl column.
A simple, sensitive, and reproducible quantitative liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was designed for the simultaneous quantification of monosaccharides derived from glycoprotein and blood serum using a multiple-reaction monitoring (MRM) approach. Sialic acids and neutral monosaccharides were efficiently separated using an amino-bonded silica phase column. Neutral monosaccharide molecules were detected as their aldol acetate anion adducts [M + CH(3)CO(2)](-) using electrospray ionization in negative ion MRM mode, while sialic acids were detected as deprotonated ions [M-H](-). The new method did not require a reduction step, and exhibited very high sensitivity to carbohydrates with limits of detection of 1 pg for the sugars studied. The linearity of the described approach spanned over three orders of magnitude (pg to ng). The method was validated for monosaccharides originating from N-linked glycans attached to glycoproteins and glycoproteins found in human blood serum. The method effectively quantified monosaccharides originating from as little as 1 microg of glycoprotein and 5 microL of blood serum. The method was robust, reproducible, and highly sensitive. It did not require reduction, derivatization or postcolumn addition of reagents.
Hammad
Loubna A
LA
METACyt Biochemical Analysis Center, Department of Chemistry, Indiana University, Bloomington, IN 47405, USA.
Derryberry
Dakota Z
DZ
Jmeian
Yazen R
YR
Mechref
Yehia
Y
D000818:Animals; D001798:Blood Proteins / Q000032:analysis; D002417:Cattle; D002853:Chromatography, Liquid / Q000295:instrumentation / Q000379:methods*; D006023:Glycoproteins / Q000032:analysis; D006801:Humans; D013058:Mass Spectrometry / Q000295:instrumentation / Q000379:methods*; D009005:Monosaccharides / Q000032:analysis*
D001798:Blood Proteins; D006023:Glycoproteins; D009005:Monosaccharides