Conjugating oligosaccharides to proteins by squaric acid diester chemistry: rapid monitoring of the progress of conjugation, and recovery of the unused ligand.
Samples that are periodically withdrawn from the mixture of a conjugation reaction can be analyzed on a picomolar scale without any work-up or pre-purification using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) in combination with the ProteinChip System. The technique provides rapid information about the increasing molecular mass of the glycoconjugate formed, thereby allowing termination of the process when the desired incorporation of the ligand onto the carrier protein is achieved. The excess oligosaccharide used at the onset of conjugation can be recovered and used in preparation of a similar neoglycoconjugate. The overall economy of conjugations, which often involve labor-intensive linker-equipped oligosaccharides, can be markedly increased in this way.
NIDDK, National Institutes of Health, Bethesda, MD 20892, USA.
A
A
Chernyak
A
A
Karavanov
Y
Y
Ogawa
P
P
Kovác
D000818:Animals; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002417:Cattle; D003503:Cyclobutanes / Q000737:chemistry*; D004952:Esters / Q000737:chemistry; D006023:Glycoproteins / Q000138:chemical synthesis*; D006031:Glycosylation; D008024:Ligands; D008969:Molecular Sequence Data; D008970:Molecular Weight; D019906:Nuclear Magnetic Resonance, Biomolecular; D019081:O Antigens / Q000737:chemistry; D009844:Oligosaccharides / Q000378:metabolism*; D012710:Serum Albumin, Bovine / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014734:Vibrio cholerae / Q000737:chemistry
D003503:Cyclobutanes; D004952:Esters; D006023:Glycoproteins; D008024:Ligands; D019081:O Antigens; D009844:Oligosaccharides; D012710:Serum Albumin, Bovine; C030042:squaric acid