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).