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.
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.
Kakehi
K
K
Faculty of Pharmaceutical Sciences, Kinki University, Higashi-osaka, Japan.
Suzuki
S
S
Honda
S
S
Lee
Y C
YC
D000581:Amidohydrolases / Q000378:metabolism; D002240:Carbohydrate Sequence; D002851:Chromatography, High Pressure Liquid; D006023:Glycoproteins / Q000737:chemistry* / Q000378:metabolism; D006834:Hydrazines / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D019158:N-Acetylneuraminic Acid; D009439:Neuraminidase / Q000378:metabolism; D009844:Oligosaccharides / Q000737:chemistry* / Q000302:isolation & purification; D010047:Ovalbumin / Q000737:chemistry / Q000378:metabolism; D010084:Oxidation-Reduction; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D011720:Pyrazoles / Q000737:chemistry*; D047069:Pyrazolones; D012260:Ribonucleases / Q000737:chemistry / Q000378:metabolism; D012794:Sialic Acids / Q000737:chemistry*; D013954:Thyroglobulin / Q000737:chemistry / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism; D000509:alpha-Fetoproteins / Q000737:chemistry / Q000378:metabolism
D006023:Glycoproteins; D006834:Hydrazines; D009844:Oligosaccharides; D011720:Pyrazoles; D047069:Pyrazolones; D012794:Sialic Acids; D000509:alpha-Fetoproteins; C029424:hydrazine; C074355:1-(4-methoxy)phenyl-3-methyl-5-pyrazolone; D010047:Ovalbumin; D013954:Thyroglobulin; D012260:Ribonucleases; C029600:ribonuclease B; D009439:Neuraminidase; D014357:Trypsin; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D019158:N-Acetylneuraminic Acid