Tandem Mass Tag-based Quantitative Proteomics Analysis To Identify Tear Fluid Biomarkers Of Perennial Allergic Conjunctivitis In Children

Purpose: Perennial Allergic Conjunctivitis is one of the most common forms of ocular allergies in children,significantly impairing patients’ quality of life.The study aimed to analyze peptide profiles in human tears using TMT quantitative proteomics to elaborate compositional differences between healthy subjects and patients suffering from PAC in children.Methods: Tear samples were collected from health and PAC children by using Schirmer tear test strips.Furthermore,digested by filter-aided sample preparation(FASP),then labeled using TMT reagent.LC-MS/MS analysis was achieved on a Q-Exactive mass spectrometer.MS/MS spectra were searched using the MASCOT engine embedded into Proteome Discoverer 1.4.Furthermore,PRM analysis was performed on target proteins using Q-Exactive HP.Skyline 3.7.0 was used for data analysis.Results: A large number of Spectra examined.Unique peptides were 8011,and 1697 protein groups were successfully identified.Statistical analysis showed that a total of 284 proteins was differentially expressed between the two groups,with 155 proteins up-regulated and 129 proteins down-regulated in PAC patients.12 target peptides of 5 identified proteins were quantitatively analyzed by PRM for absolute quantification.Tear levels of EPO(P=0.01),ECP(P=0.01),MBP(P=0.02),and LEG7(P<0.01)showed significant differences in PAC patients compared to controls.Conclusion: Proteomic analysis with TMT is a powerful tool for screening large numbers of proteins in tears.Eosinophils play a pathophysiologic role in allergic inflammation.ECP,EPO,and MBP are essential biomarkers in PAC.Moreover,it revealed previously undetected proteins-LEG7 that might participate in the pathogenesis of PAC.