TEAR FLUID N-GLYCAN PROFILING TO INVESTIGATE BIOMARKERS IN VERNAL AND ATOPIC KERATOCONJUNCTIVITIS

Abstract

Purpose: Proteomics and glycogene-chips studies revealed that several tear proteins are glycosylated. Glycoproteins play a role in different function of the ocular surface including protection against pathogens, immunoregulation and prevention of desiccation. In the present study, we analyze the N-linked glycome of tear fluid from patients affected by vernal (VKC) and atopic keratoconjunctivitis (AKC) in order to identify potential biomarkers for these diseases. To date, no specific laboratory test are suitable for VKC and AKC diagnosis and monitoring.

Methods: Tear samples from 23 VKC patients, 7 AKC patients and 11 control subjects were diluted 1/10 in a denaturant solution, reduced, alkylated and treated with N-glycosidase F (PNGase F), an enzyme which specifically deglycosylates N-glycoproteins. Released N-glycans were purified, chemically derivate by permethylation and analyzed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS and MALDI TOF/TOF MS/MS). The eligibility of mass spectrometry for the study of glycosylation is due to its high sensibility and ability to analyze complex mixtures of glycans from biological samples.

Results: Our approach allowed to identify more than 150 complex N-glycans, including biantennary, triantennary, tetraantennary and bisecting species. Highly fucosylated structures were also found. Data analysis showed changes in terms of relative intensities for some structures. In VKC, the most significant peaks were related to mass to charge ratios 1906.7 and 2592.3, corresponding to a bisecting and to a N-glycan structure bearing two Lewis-X epitope, respectively.In AKC the most intense peak is found at m/z 2792.4, and corresponds to the well-known biantennary, disialylated N-glycan. Each structure reveals significant differences between healthy and pathological conditions, with major variations involving bisecting and hyperfucosylated glycoforms. Structures were confirmed in detail by MS/MS analysis.

Conclusions: MALDI-TOF-MS is a valuable technique for biomarker detection and characterization of defective glycan structures. Identified peaks could be used as potential biomarkers for VKC and AKC. Further structural characterization of the disease-associated N-glycans may confirm these preliminary results and allow to discover additional biomarkers.