Figure 2
Mass spectrometric analysis of N-linked glycans from GalMBP ligands on L-428 cells. Purified GalMBP ligands were digested with trypsin and N-linked glycans were released by peptide:N-glycosidase F. N-linked glycans were permethylated and subjected to Sep-Pak purification. A. The MALDI MS spectrum of one of the elution fractions from the Sep-Pak is shown. All labeled ions were subjected to MS/MS analysis. The schemes for each ion represent the most likely structure based on the fit between the calculated composition and the m/z (z = 1) ratio of the ions detected, taking into account the biosynthetic pathways of N-glycosylation in addition to the MS and MS/MS data collected. Structures in red boxes contain terminal fucose residues. B. An example of the MALDI MS/MS spectra, showing fragmentation of the 2591 molecular ion. The presence of major fragment ions at 1954 and 660 confirms the presence of terminal Lewisx or Lewisa structures. Specific evidence for the presence of Lewisx structures is provided by elimination of fucose from the 3 position of GlcNAc to form the 2385 fragment ion. The presence of unfucosylated glycans that are suboptimal ligands for GalMBP, as well as high-mannose oligosaccharides that do not bind GalMBP at all, is presumed to reflect the presence of heavily glycosylated glycoproteins that also contain complex-type glycans bound by GalMBP as previously observed for breast cancer cells [6].