N-glycosylation analysis of control tear fluid
Tear samples from the 11 CTRL were processed as described and analyzed by MALDI TOF MS in order to define the typical tear N- glycan pattern in healthy subjects. Permethylated glycans were detected in positive polarity as Na+ ion adducts, whose molecular masses differ from their theoretical protonated counterpart by 22 u (atomic mass unit). All the CTRL mass spectra were found very similar to the profile in Figure 1A, showing more than 150 peaks corresponding to [M + Na]+ pseudo-molecular ions from m/z1579.8 to m/z 4432.2 (supplementary Table 2). Spectra showed the occurrence of a wide range of N- glycans categories including oligomannose, complex bi-, tri-, and tetra-antennary, neutral, acidic, bisected and hybrid species. Additionally, the main peaks were further analyzed by MALDI TOF/TOF MS/MS. This analysis was crucial to discriminate, in most cases, between isomeric glycoforms or to confirm the presence of two or even more isomeric glycoforms associated to a single molecular mass.