Mono-, di- and multi-fucosylated N-glycans
Amongst the N- glycan structures (approximately 150) listed in supplementary Table 2, 116 (about 75%) were fucosylated, being 41 out 116 mono-fucosylated and 75 out 116 bearing more than one fucose unit, with two glycans having up to seven fucose moieties. Core fucosylated and peripheral fucosylatedN- glycans, depending on fucose localization, were both present in the CTRL tear N- glycome profile. In the present study, core fucosylation (due to an α1-6 fucose linked to theN- acetylglucosamine bound to the protein), and peripheral fucosylation, (due to a fucose moiety 1-3 or 1-4 linked to an antennary N-acetylglucosamine or 1-2 linked to a galatose [32]) were characterized by the occurrence of specific MS/MS ions. Antennary fucosylation allowed us to recognize specific epitopes belonging to the Lewis family (structural features typical of blood group antigens [33]) (see supplementary Table 3).
Corefucosylated glycans and/or N- glycans bearing Lex/a, sLex/a or Ley/b motifs were identified (current approaches based MALDI MS and MS/MS on permethylated N- glycans do not provide informations on linkage positions, in order to discriminate (s)Lea from (s)Lex and Leb from Ley).
The occurrence of a Lex/a epitope was confirmed by the ion at m/z 660.3 in the fragmentation spectra of the glycan strucures at m/z 2418.2, 2592.3, 2605.3, 2766.4 and 2779.4. Figure 2 shows the MS/MS spectrum of the latter peak, whereas the other fragmentation spectra are reported in the supplementary (supplementary Figures S5, S6, S7, S8). MS/MS analysis allowed us to identify some ions corresponding to more than one isomeric structure (i.e., peak atm/z 2605.3 in Figure S7 is actually related to a mixture of two glycans, a corefucosylated one and a second bearing a Lex/a epitope).
The sLex/a motif, recognized through the fragment atm/z 1021.5, was associated to the glycan structures at m/z3127.6, 3140.6 and 3589.8 (supplementary Figures S9, S10, S11). Interstingly, the structure at m/z 3127.6 corresponded to a glycoform owning four fucoses, suggesting that corefucose, sLex/a and Ley/b epitopes are all present in the same glycan (supplementary Figure 9).
Ley (and/or Leb) pattern is proven by the fragment at m/z 834.4 occurring in the fragmentation spectra of the glycans at m/z 2418.2, 2592.3, 2766.4, 2940.5 and 3127.6 (see supplementary Figures S5, S6, S8, S12 and S9 respectively).