| Background: Aqueous humor(AH),as an important component of the ocular circulation and immune and eye's optical system is far from being understood on a molecular scale.Major advances in mass spectrometry instrumentation,proteomic methodologies,and bioinformatics have dramatically transformed the ophthalmology and vision science within the last few years.proteomic workflows can reveal the large proteome datasets providing important function and pathway information about AH and ophthalmic diseases.Method: In this study,a pooled AH sample from 21 volunteer patients with cataract was used to provide a comprehensive profile of human AH proteome by LCMS/MS method.480 proteins in qualitative proteins were quantitatively analyzed based on the peak intensity semi quantitative method(IBAQ),and the proteins were classified according to different abundances,and the functions of proteins in different abundance regions were analyzed.The proteome of aqueous humor was further compared with plasma and other ocular fluids such as vitreous humor and tears.In the follow-up study of comparative evaluation of the aqueous humour proteome of primary acute-angle and chronic angle-closure,neovascular glaucoma and age-related cataract eyes,a total of175 subjects including 57 primary acute angle-closure glaucoma(PAACG),50 Primary chronic angle-closure glaucoma(PCACG),35 neovascular glaucoma(NVG)and 33age-and gender-matched cataract patients were enrolled and comparison proteomic analysis was performed.Above samples were randomly divided into two cohorts,discovery cohort and validation cohort,whose AH proteome were analyzed by dataindependent acquisition(DIA)method and by parallel reaction monitor(PRM)method respectively.Subsequently,the researchers examined the proteomic response of aqueous humor in patients with neovascular glaucoma(NVG)before and after treatment with conbercept.Three patients with NVG were included in the study group,and a total of 6 ah samples(3 pre-treatment samples vs 3 post-treatment samples)were compared with those before and after treatment by dia technology.Proteins with fold change FC > 1.5 were considered as differential expression proteins(DEPs).Furthermore,the interaction network of DEPs was constructed by using Cytoscape and string database.The highest nodedegree protein in the center of the network was calculated by using hubgene plug-in,and the neighbor protein with FN1 was selected for further verification.The validation group included 11 NVG patients,and 14 ah samples were collected as validation group,including 7 cases before treatment and 7cases after treatment.PRM technology was used to verify the target DEPs,and the multiple change > 1.5,and P < 0.05 were used as the screening criteria for validation proteins,and the network analysis of the final verified proteins was carried out.Results: a total of 802 proteins were identified in the aqueous humor proteome database,of which 318 proteins were identified for the first time in AH.The quantitative analysis of 480 proteins showed that the range was over 7 orders of magnitude.The results were compared with other similar studies in recent years,which showed that the protein expression in aqueous humor was relatively stable.Most of aqueous humor proteins are plasma derived proteins,which are mainly involved in immune and inflammatory pathways.The function of aqueous humor proteome with different abundance was compared.The high abundance group mainly participated in inflammatory reaction,the medium abundance group mainly involved in energy metabolism,while the low abundance group had a large number and complex functions.The proteome of aqueous humor was compared with that of plasma,vitreous fluid and tear.Most of aqueous humor proteins are common with plasma proteins(91.2% of the total mass).Aqueous humor specific proteins are mostly low abundance proteins,which are significantly enriched in taurine and 1,25-dihydroxyvitamin D3 biosynthesis pathway.Compared with the three kinds of ocular fluid,the function of aqueous humor proteome is mainly focused on different types of endocytosis,the function of vitreous fluid proteome is focused on energy metabolism,and tear protein is more involved in protein metabolism and apoptosis.In the proteome comparative study of different types of glaucoma,it was found that compared with cataract,the common molecular characteristics of the three glaucoma diseases were immune response,lipid metabolism and cell apoptosis.At the same time,the three glaucoma diseases showed different degrees of immune disorder.In glaucoma and glaucoma,ndpi1 and servtn were upregulated.The proteome analysis of PAACG,PCACG and NVG revealed the different characteristics of three kinds of glaucoma: NVG is characterized by angiogenesis activation;PAACG is characterized by inflammatory reaction activation.Serpind1 has been found to play an important role in the development of glaucoma.It is related to the decrease of optical transmittance and glucose metabolism.Through further evaluation of DEPs,it is found that the combination of SERPIND1,CARTPT and CDHR1 can distinguish different types of glaucoma.In the study of using proteome to detect the efficacy of glaucoma,the researchers identified 636 proteins in aqueous humor by DIA method before conbercept treatment,and analyzed 559 proteins quantitatively;after treatment,636 proteins and 566 proteins were identified in aqueous humor samples.The differential expression proteins(DEPs)with fold change(FC)>1.5 were verified by gene ontology analysis and path analysis.Finally,26 differentially expressed proteins were screened out.Through gene ontology analysis,it was found that the functions of DEPs were mainly focused on binding and catalytic activities.Further analysis of IPA function indicates that conbercept treatment mainly interferes with angiogenesis and coagulation.It is worth noting that,combined with the results of the previous chapters,the researchers found that the lipid metabolism may an active participant in the pathogenesis and treatment of various types of glaucoma.This part of the study shows that AH proteomics can reflect a variety of physiological and pathological changes in the process of glaucoma treatment.Conclusion: AH proteome is an important potential source of biomarkers for identifying posterior pathophysiological changes and can provide a basis for observing the therapeutic effect.This study will provide insights in understanding molecular mechanism of types of glaucoma and contribute to the application of AH proteome in the diagnosis and treatment of glaucoma ophthalmic disease. |
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