Investigation of cationic peanut peroxidase glycans by electrospray ionization mass spectrometry.
Cationic peanut peroxidase (CP) was isolated from peanut (Arachis hypogaea) cell suspension culture medium. CP is a glycoprotein with three N-linked glycan sites at Asn60, Asn144, and Asn185. ESI-MS of the intact purified protein reveals the microheterogeneity of the glycans. Tryptic digestion of CP gave a near complete sequence coverage by ESI-MS. The glycopeptides from the tryptic digestion were separated by RP HPLC identified by ESI-MS and the structure of the glycan chains determined by ESI-MS/MS. The glycans are large structures of up to 16 sugars, but most of their non-reducing ends have been modified giving a mixture of shorter chains at each site. Good agreement was found with the one glycan previously analyzed by (1)H NMR. This work is the basis for the future studies on the role of the glycans on stability and folding of CP and is another example of a detailed structural characterization of complex glycoproteins by mass spectrometry.
Zhang
Cunjie
C
Department of Biochemistry, The University of Western Ontario, London, Ont., Canada N6A 5C1.
Doherty-Kirby
Amanda
A
Huystee Rv
Robert van
Rv
Lajoie
Gilles
G
D010367:Arachis / Q000737:chemistry*; D002236:Carbohydrate Conformation; D002851:Chromatography, High Pressure Liquid; D003470:Culture Media; D009195:Peroxidase / Q000737:chemistry*; D011134:Polysaccharides / Q000737:chemistry*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
D003470:Culture Media; D011134:Polysaccharides; D009195:Peroxidase