TY - JOUR
T1 - Multiple-Reaction Monitoring Liquid Chromatography Mass Spectrometry for Monosaccharide Compositional Analysis of Glycoproteins
AU - Hammad, Loubna A.
AU - Saleh, Marwa M.
AU - Novotny, Milos V.
AU - Mechref, Yehia
N1 - Funding Information:
The authors acknowledge support for this work primarily by the Indiana Metabolomics and Cytomics Initiative (METACyt), funded by a grant from Eli Lilly Endowment. The authors also acknowledge partial support for this work by grant no. RR018942 from the National Center for Research Resources, a component of the National Institute of Health (NIH-NCRR) for the National Center for Glycomics and Glycoproteomics (NCGG) at Indiana University.
PY - 2009/6
Y1 - 2009/6
N2 - 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 + CH3CO2]- 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 μg of fetuin, ribonuclease B, peroxidase, and α1-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.
AB - 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 + CH3CO2]- 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 μg of fetuin, ribonuclease B, peroxidase, and α1-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.
UR - http://www.scopus.com/inward/record.url?scp=65549158689&partnerID=8YFLogxK
U2 - 10.1016/j.jasms.2009.02.022
DO - 10.1016/j.jasms.2009.02.022
M3 - Article
C2 - 19318280
AN - SCOPUS:65549158689
VL - 20
SP - 1224
EP - 1234
JO - Journal of the American Society for Mass Spectrometry
JF - Journal of the American Society for Mass Spectrometry
SN - 1044-0305
IS - 6
ER -