Characterization of oligosaccharide moieties of intact glycoproteins by microwave-assisted partial acid hydrolysis and mass spectrometry.
A method, which utilizes microwave-assisted partial acid hydrolysis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS), to elucidate oligosaccharide composition of intact glycoproteins is presented here. Glycoproteins, such as ribonuclease B, avidin, alpha1-acid glycoprotein, and fetuin, are used as model systems to demonstrate this technique. Partial cleavage of oligosaccharides from whole intact glycoproteins with trifluoroacetic acid was observed after a short exposure to microwaves. Due to the high-resolution mass spectra obtained by MALDI-TOFMS from glycoproteins with molecular weights less than 20 kDa, the compositions of oligosaccharides are readily derived for ribonuclease B and avidin. The data agree with the proposed oligosaccharide structures of ribonuclease B (five glycoforms) and avidin (eight glycoforms). Larger glycoproteins such as alpha1-acid glycoprotein (many glycoforms) and fetuin (many glycoforms) exhibited only broad peaks with no glycoform resolution. Nevertheless, this method can be used successfully for analysis of glycoproteins with molecular weights greater than 20 kDa to determine the presence or absence of glycosylation.
Lee
Bao-Shiang
BS
Protein Research Laboratory, Research Resources Center, 835 S. Wolcott Avenue, University of Illinois, Chicago, IL 60612, USA. boblee@uic.edu
Krishnanchettiar
Sangeeth
S
Lateef
Syed Salman
SS
Lateef
Nabila Salman
NS
Gupta
Shalini
S
D000143:Acids / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D001360:Avidin / Q000737:chemistry; D004529:Egg White; D006023:Glycoproteins / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008872:Microwaves; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000032:analysis* / Q000737:chemistry*; D012260:Ribonucleases / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D000143:Acids; D006023:Glycoproteins; D009844:Oligosaccharides; D001360:Avidin; D012260:Ribonucleases; C029600:ribonuclease B