Figure 2. The N-glycome of axenically-cultured Dicytostelium discoideum.

(A) MALDI-TOF MS of pyridylaminated N-glycans from axenically-grown mid-log phase AX3 either released from glycopeptides using PNGase A, PNGase F or EndoH or from intact proteins treated with PNGase F showing the absence of fucosylated glycans from the latter three glycan pools. The peaks marked with an asterisk in the spectrum of the EndoH-released glycans are non-pyridylaminated forms of the Hex₉HexNAc₁ (m/z 1703) and Hex₈HexNAc₃ (m/z 1947) species. The data with pyridylaminated PNGase A-released glycans is compatible to those of the unlabelled, free N-glycans (see ESI-MS data in Supplementary Figure 2). (B) MALDI-TOF MS of PNGase A-released pyridylaminated N-glycans from axenically-grown mid-log phase HL250 grown in the absence or presence of exogenously-added fucose, showing the appearance of a fucosylated H8N4F glycan (m/z 2373) in the latter. C) RP-HPLC pyridylaminated PNGase A-released N-glycans from axenically-grown mid-log phase AX3 and HL250 grown in the absence of exogenous fucose shows a difference in elution properties; peaks were collected and subject to MALDI-TOF MS (major species are annotated) and compared to an isomaltooligosaccharide standard (g.u., glucose units). The major [M+Na]⁺ species are annotated in the form H_xN_yF_0-1, where H signifies hexose, N is N-acetylhexosamine and F is fucose.