Glycoconjugate analysis of hGGT1 expressed in Pichia pastoris . (A) MALDI-TOF-MS profile of neutral N-glycans released by PNGase F from purified Pp-hGGT1. Ions corresponding to monosodiated N-glycans known to be expressed by Pichia pastoris are assigned. Symbols: blue box, GlcNAc; green circle, mannose. (B) Averaged LC/Q-Star MS spectrum for the Asn-120 (LAFATMFNSSEQSQK) family of glycopeptides from Pp-hGGT1, showing the identified glycoconjugates within their elution interval. Asterisks denote glycoconjugates that were confirmed by tandem MS analysis. P represents the LAFATMFNSSEQSQK tryptic peptide to which the N-glycans are attached. Unannotated peaks represent co-eluting nonglycosylated hGGT1 peptides. (C) MS/MS spectrum showing fragmentation of the Man11GlcNAc2-modified glycopeptide (m/z =1293.2) shown in (B).