Sample preparation
8 µL of tears from CTRL, VKC and AKC patients were diluted 1/10 in a 0.1
% denaturant solution of RapiGest™ (Waters Corporation, Milford,
Massachusetts) in 50 mM NH4HCO3.
Denaturated (glyco)proteins were reduced by adding 100 mM dithiothreitol
(DTT) in 50 mM NH4HCO3 (final
concentration 5 mM) and incubated for 30 min at 56°C. Subsequently,
cysteine alkylation was achieved by an addition of 100 mM iodoacetic
acid (IAA) in 50 mM NH4HCO3 (final
concentration 15 mM) and incubation for 40 min at room temperature in
the dark. The N- linked glycans were enzymatically released by the
action of peptide-N-glycosidase F (PNGase F, from Flavobacterium
meningosepticum EC 3.5.1.52; Roche Diagnostics GmbH) (2 ml corresponding
to 2 units) at 37°C overnight and then purified by solid-phase
extraction using Hypercarb cartridges (Thermo Fisher Scientific,
Bellefonte, USA). PNGase F treatment is higly efficient in these
conditions [28].
Glycan permethylation was afforded as previously described [29]
according to Ciucanu and Kerek protocol [30], to further enhance
detection sensitivity upon MS investigation. PermethylatedN- glycans were then subjected to MS investigation by MALDI TOF MS
and MALDI TOF/TOF MS/MS to achieve a detailed structural elucidation of
some N- glycans and univocally establish their structures.