Characterization of the glycosylation of recombinantEndopolygalacturonase I fromAspergillus niger

Abstract

The carbohydrate chains of recombinant endopolygalacturonase I (EPG I) from Aspergillus niger were characterized using a combination of mass spectrometric techniques. High performance liquid chromatography (HPLC) in conjunction with electrospray ionization mass spectrometry was used to separate the components of EPG I liberated by trypsin digestion. In-source collision-induced dissociation (CID) was utilized to fragment the digestion products entering the mass spectrometer, and the generation of carbohydrate fragment ions allowed for the identification of glycopeptides. The masses of the resulting glycans were calculated and entered into a carbohydrate database to search for possible structures. The primary sequences of the carbohydrate chains were confirmed by digesting aliquots of the intact glycopeptide with endo- and exoglycosidases and then analyzing the digestion products using matrix-assisted laser desorption/ionization mass spectrometry. These experiments demonstrated that one of the two N-linked sites of EPG I was occupied by a series of high-mannose structures, the second N-linked site was not occupied, and no O-linked sites were detected.

Post‐translational modifications of recombinant B. cinerea EPG 6

“…In this work BcPG6 from the B. cinerea strain B05.10 was over‐expressed in Pichia pastoris , and the glycosylation sites and attached oligosaccharide structures were analyzed. The observation of multiple N ‐linked glycosylation signals suggested that BcPG6 is produced as a glycosylated protein, as previously observed for EPGs produced from the fungi A. niger 14, 15 and S. purpureum 16. The molecular mass of BcPG6 was identified by matrix‐assisted laser desorption/ionization mass spectrometry (MALDI‐MS), and was shown to have a significantly higher molecular mass than that calculated from its amino acid sequence, a further indication of post‐translational modification (PTM).…”

On-Target Exoglycosidase Digestions/MALDI-MS for Determining the Primary Structures of Carbohydrate Chains

“…These digests have been proven successful for oligosaccharides of known structures, and we wanted to test their success as aids in sequencing carbohydrate chains of unknown structures. LC/MS analysis of trypsin-digested pectate lyase from A. niger identified one fraction that contained a glycopeptide . Using mass information obtained from the LC/MS experiment and other sequence information, the mass of the carbohydrate was calculated.…”

“…Pectate lyase from Aspergillus niger was a gift from Jaap Visser at Wageningen Agricultural University, Wageningen, The Netherlands. The glycopeptide from trypsin-digested pectate lyase was previously isolated and identified using liquid chromatography/mass spectrometry (LC/MS) . The fraction containing the glycopeptide was dried and reconstituted with water to a concentration of ∼10 μM.…”

On‐target endoglycosidase digestion matrix‐assisted laser desorption/ionization mass spectrometry of glycopeptides

“…Fractions were manually collected every 2 min during the gradient. Additional details on this procedure can be obtained from the literature 13–15. The LC fractions identified as containing N ‐linked glycans were used for the endo glycosidase digestions.…”