Proteomic Study of Aqueous Humor and Its Application in the Treatment of Neovascular Glaucoma

Abstract

Aqueous humor (AH) proteins are involved in many physiological and pathological processes of the eye. The proteome analysis of AH is important to understand its physiological and pathophysiological functions. In the present study, AH samples obtained from 21 cataract volunteers were pooled together. After high-pH RPLC offline separation, the pooled sample was analyzed by LC-MS/MS to provide a comprehensive profile of AH proteome. The function analysis was provided by the GO and IPA annotation. In order to determine whether the AH proteome can reflect the pathophysiological changes of the disease, DIA technology was used to analyze the AH samples obtained from three neovascular glaucoma (NVG) patients (six samples) before and after drug treatment. The differential proteins were validated by PRM technology in an independent group (14 samples). In the AH proteome database, 802 proteins were identified, and 318 proteins were identified for the first time. Furthermore, 480 proteins were quantified based on the peak intensity-based semiquantification (iBAQ), which ranged by approximately 7 orders of magnitude. These proteins are primarily involved in immunity- and inflammation-related pathways. The differential AH proteomic analysis in NVG treatment revealed that the AH proteome can reflect the pathophysiological changes of drug treatment. Angiogenesis and thrombus coagulation progression are deeply involved in NVG treatment. The present experiment provided a comprehensive AH proteome analysis and expanded the profile of human AH proteome. The differential AH proteomic analysis of NVG treatment indicated that AH proteome can reflect the pathophysiological changes in drug intervention.

Keywords: aqueous humor; conbercept; neovascular glaucoma treatment; proteome; vascular endothelial growth factor receptor.

FIGURE 1

Workflow of the human aqueous humor (AH) fluid proteome profile analysis and the discovery/validation of differently expressed proteins (DEPs) in the neovascular glaucoma (NVG) treatment.

FIGURE 2

The AH proteome analysis of the present study. (A) The Venn diagram of the identified proteins and comparison of the present results with the previous AH proteome data. (B) The Ingenuity Pathway Analysis (IPA) canonical pathway analysis of the AH proteome. (C) The hierarchical clustering based on the intensity-based absolute quantification (iBAQ) quantification and IPA annotation. (D) The dynamic range of the AH proteome with key biomarkers marked. The X-axis indicates the protein numbers ranked by the iBAQ intensity, and the Y-axis indicates the log10 (relative intensity) of the proteins.

FIGURE 3

The DEP analysis of the AH proteome in the NVG treatment with conbercept. (A) The orthogonal partial least squares discriminant analysis (OPLS-DA) score plot based on the DIA data of the AH proteome from NVG patients (green), and after conbercept treatment patients (blue). The mean center-scaling model (P = 1.54857e-07). (B) The IPA ingenuity canonical pathway analysis of DEPs. (C) The subnetwork of DEPs with high node degree. (D) The hierarchical clustering based on the PRM data of the validated DEPs. (E) The function annotation of validated DEPs. (F) The network of validated proteins.

FIGURE 4

Comparison of the AH and body fluid proteome. (A) The pathway comparison between AH-specific proteins and plasma-derived proteins. (B) The pathway comparison between AH, VH, and tears. (C) The functional comparison between AH, VH, and tears.