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.