New insights in Rapana venosa hemocyanin N-glycosylation resulting from on-line mass spectrometric analyses.
The N-glycosylation of structural unit 1 of Rapana venosa hemocyanin was studied. Enzymatically liberated N-glycans were analyzed by matrix-assisted laser desorption ionization-time-of-flight-mass spectrometry (MALDI-TOF-MS) and capillary electrophoresis (CE)-MS following 8-aminopyrene-1,3,6-trisulfonate labeling and labeling with 3-aminopyrazole, a new dedicated sugar reagent. Structural information was obtained by exoglycosidase sequencing, on-line MS/MS, permethylation, and amidation. A mixture of high-mannose and complex glycans with so far unknown and unusual acidic terminal structures was revealed. As the hemocyanin protein sequence is currently unknown, de novo sequencing of the glycopeptides had to be carried out. The N-glycans were therefore enzymatically removed with simultaneous partial (50%) (18)O-labeling of glycosylated asparagine residues prior to proteolysis. Following nano-liquid chromatography-MALDI-TOF-MS, the originally glycosylated peptides could be revealed and their sequences determined by MS/MS. The site occupancies were subsequently elucidated by precursor ion scanning of the intact glycopeptides using a Q-Trap mass spectrometer.
Sandra
Koen
K
Laboratory of Protein Biochemistry and Protein Engineering, Ghent University, KL Ledeganckstraat 35, B-9000 Ghent, Belgium. koen.sandra@peakadilly.com
Dolashka-Angelova
Pavlina
P
Devreese
Bart
B
Van Beeumen
Jozef
J
D000595:Amino Acid Sequence; D000818:Animals; D019075:Electrophoresis, Capillary; D049851:Gastropoda / Q000378:metabolism*; D006026:Glycoside Hydrolases / Q000737:chemistry; D006031:Glycosylation; D006433:Hemocyanins / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D009862:Online Systems; D011134:Polysaccharides / Q000032:analysis; D011720:Pyrazoles / Q000737:chemistry; D011721:Pyrenes / Q000737:chemistry; D020539:Sequence Analysis, Protein; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014357:Trypsin / Q000737:chemistry
C436945:3-aminopyrazole; C103430:8-aminopyrene-1,3,6-trisulfonic acid; D011134:Polysaccharides; D011720:Pyrazoles; D011721:Pyrenes; D006433:Hemocyanins; D006026:Glycoside Hydrolases; D014357:Trypsin