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Precolumn Labeling of Reducing Carbohydrates With 1-(p-methoxy)phenyl-3-methyl-5-pyrazolone: Analysis of Neutral and Sialic Acid-Containing Oligosaccharides Found in Glycoproteins

Precolumn Labeling of Reducing Carbohydrates With 1-(p-methoxy)phenyl-3-methyl-5-pyrazolone: Analysis of Neutral and Sialic Acid-Containing Oligosaccharides Found in Glycoproteins

K Kakehi, S Suzuki, S Honda, Y C Lee

Anal Biochem. 1991 Dec;199(2):256-68.

PMID: 1725951

Abstract:

A convenient precolumn labeling method was developed for the analysis of neutral and sialic acid-containing oligosaccharides in glycoproteins using 1-(p-methoxy)phenyl-3-methyl-5-pyrazolone (PMPMP). PMPMP reacts with a reducing oligosaccharide under slightly alkaline conditions (pH 8.3) to form a 2:1 adduct (bis-PMPMP derivative). Sialic acid residues in the oligosaccharides remain intact during the reaction. Tryptic glycopeptides digested with glycopeptidase A for oligosaccharide liberation can be directly derivatized with PMPMP without prior treatment. Separation of the labeled oligosaccharides was performed by reverse-phase high-performance liquid chromatography on a C-18 column with aqueous acetonitrile, and positional isomers such as isomeric triantennary tetradecasaccharides from bovine fetuin were completely resolved. The bis-PMPMP derivatives were labile in alkaline media to form mono-PMPMP derivatives; however, the mono-PMPMP derivatives could be easily reconverted to the original bis-PMPMP derivatives. The proposed method is simpler than the reductive pyridylamination method, and detection sensitivity could reach subnanomole range with a uv detector. Oligosaccharides from ribonuclease B (bovine pancreas), ovalbumin, thyroglobulin (porcine thyroid), fetuin (bovine), and transferrin (human) have been successfully analyzed to demonstrate the usefulness of this method as an alternative to the existing methods.

Chemicals Related in the Paper:

Catalog Number	Product Name	Structure	CAS Number	Price
IAR42412298	Trypsin Acetylated from bovine pancreas			Price

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