Direct analysis of enzymatic reactions of oligosaccharides in human serum using matrix-assisted laser desorption ionization mass spectrometry.
Matrix-assisted laser desorption ionization mass spectrometry has been developed for direct mass analysis of enzymatic reaction products of oligosaccharides in human blood serum without the use of extraction or chromatographic separation. Molecular labeling of the substrate is used to achieve both the detection sensitivity and selectivity required in rapid analysis of reaction products in serum. It is found that tetramethylrhodamine (TMR)-labeled oligosaccharides provide 100-fold sensitivity enhancement over the corresponding underivatized oligosaccharides. In order to selectively retain the TMR-labeled molecules on the sample probe while washing away contaminants in a serum sample, a sample/matrix preparation method is developed. This technique provides detection sensitivity of labeled oligosaccharides in the range of hundreds of femtomoles per microliter. The mass measurement accuracy is better than 0.01% when a linear time-of-flight mass spectrometer is used. The application of the technique is illustrated for the subpicomole detection and quantitation of the conversion of the disaccharide alpha Fuc(1-->2)beta Gal-TMR to the blood group B active trisaccharide alpha Fuc(1-->2)[alpha Gal(1-->3)]beta Gal-TMR, catalyzed by the blood group B galactosyltransferase present in human serum.
Whittal
R M
RM
Department of Chemistry, University of Alberta, Edmonton, Canada.
Palcic
M M
MM
Hindsgaul
O
O
Li
L
L
D004798:Enzymes / Q000737:chemistry; D006801:Humans; D009844:Oligosaccharides / Q000097:blood*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D004798:Enzymes; D009844:Oligosaccharides