title: Multiple-reaction monitoring liquid chromatography mass spectrometry for monosaccharide compositional analysis of glycoproteins.
abstract: A simple, sensitive, and rapid quantitative LC-MS/MS assay was designed for the simultaneous quantification of free and glycoprotein bound monosaccharides using a multiple reaction monitoring (MRM) approach. This study represents the first example of using LC-MS/MS methods to simultaneously quantify all common glycoprotein monosaccharides, including neutral and acidic monosaccharides. Sialic acids and reduced forms of neutral monosaccharides are efficiently separated using a porous graphitized carbon column. Neutral monosaccharide molecules are detected as their alditol acetate anion adducts [M + CH(3)CO(2)](-) using electrospray ionization in negative ion MRM mode, while sialic acids are detected as deprotonated ions [M - H](-). The new method exhibits very high sensitivity to carbohydrates with limits of detection as low as 1 pg for glucose, galactose, and mannose, and below 10 pg for other monosaccharides. The linearity of the described approach spans over three orders of magnitudes (pg to ng). The method effectively quantified monosaccharides originating from as little as 1 microg of fetuin, ribonuclease B, peroxidase, and alpha(1)-acid glycoprotein human (AGP) with results consistent with literature values and with independent CE-LIF measurements. The method is robust, rapid, and highly sensitive. It does not require derivatization or postcolumn addition of reagents.
authors.affiliation: METACyt Biochemical Analysis Center, Department of Chemistry, Indiana University, Bloomington, Indiana 47405, USA.
authors.forename: Loubna A
authors.initials: LA
authors.lastname: Hammad
authors.forename: Marwa M
authors.initials: MM
authors.lastname: Saleh
authors.forename: Milos V
authors.initials: MV
authors.lastname: Novotny
authors.forename: Yehia
authors.initials: Y
authors.lastname: Mechref