Identification of the glycosylation site and glycan structures of recombinant endopolygalacturonase II by mass spectrometry.
A series of mass spectrometric experiments was performed to characterize the carbohydrate chains attached to endopolygalacturonase II (EPG-II) overexpressed in Aspergillus niger. First, an aliquot of trypsin-digested EPG-II was analyzed by capillary high-performance liquid chromatography (HPLC) coupled with electrospray ionization (ESI) mass spectrometry (HPLC/MS). The ESI mass spectrometer was operated in the tandem mass spectrometric (MS/MS) mode and utilized precursor ion scanning of the HexNAc+ oxonium ion at m/z 204 to selectively detect glycopeptides eluting from the HPLC. Aliquots of the fraction identified by HPLC/MS/MS to contain a glycopeptide were then sequentially digested with Aspergillus saitoi alpha-1-2-mannosidase and peptide N:glycosidase F (N-glycanase), followed by matrix-assisted laser-desorption/ionization mass spectrometric analysis to provide structural information from the carbohydrate chain. The masses of the carbohydrate chains in the glycopeptides were thus determined and subsequently used to search the Complex Carbohydrate Structure Database for the probable structures of the glycans. This analysis determined that recombinant EPG-II has a single N-glycosylation site, the carbohydrate chain at this site is heterogeneous, and the glycan structure is of the high-mannose type. No sites of O-glycosylation were detected in EPG-II.
Yang
Y
Y
Complex Carbohydrate Research Center, University of Georgia, Athens 30602-4712, USA.
Bergmann
C
C
Benen
J
J
Orlando
R
R
D000595:Amino Acid Sequence; D001234:Aspergillus niger / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid; D006031:Glycosylation; D007527:Isoenzymes / Q000737:chemistry*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011096:Polygalacturonase / Q000737:chemistry*; D011134:Polysaccharides / Q000737:chemistry*; D011994:Recombinant Proteins / Q000032:analysis; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D007527:Isoenzymes; D011134:Polysaccharides; D011994:Recombinant Proteins; D011096:Polygalacturonase