The Constitutive Proteome of Human Aqueous Humor and Race Specific Alterations

Abstract

Aqueous humor (AH) is the fluid in the anterior and posterior chambers of the eye that contains proteins regulating ocular homeostasis. Analysis of aqueous humor proteome is challenging, mainly due to low sample volume and protein concentration. In this study, by utilizing state of the art technology, we performed Liquid-Chromatography Mass spectrometry (LC-MS/MS) analysis of 88 aqueous humor samples from subjects undergoing cataract surgery. A total of 2263 unique proteins were identified, which were sub-divided into four categories that were based on their detection in the number of samples: High (n = 152), Medium (n = 91), Low (n = 128), and Rare (n = 1892). A total of 243 proteins detected in at least 50% of the samples were considered as the constitutive proteome of human aqueous humor. The biological processes and pathways enriched in the AH proteins mainly include vesicle mediated transport, acute phase response signaling, LXR/RXR activation, complement system, and secretion. The enriched molecular functions are endopeptidase activity, and various binding functions, such as protein binding, lipid binding, and ion binding. Additionally, this study provides a novel insight into race specific differences in the AH proteome. A total of six proteins were upregulated, and five proteins were downregulated in African American subjects as compared to Caucasians.

Keywords: aqueous humor; mass spectrometry; proteomics.

Figure 1

Liquid chromatography/Mass Spectrometry (LC-MS/MS) workflow for proteomic analysis of human aqueous humor. Samples were digested using trypsin and were analyzed using an Orbitrap Fusion Tribrid mass spectrometer coupled with an Ultimate 3000 nano-UPLC system. Proteins were identified and quantified using Proteome Discoverer (ver 1.4; Thermo Scientific, Waltham, MA, USA) followed by statistical analysis using the R Project for statistical computing (https://www.r-project.org/).

Figure 2

Example of LC-MS/MS analysis of human aqueous humor sample. (A) LC-MS/MS total ion current chromatogram. The retention time (RT) elution of one reporter peptide indicative of A2M protein is marked for illustration purposes. (B) MS spectra of selected precursor peptide 444.78 m/z with a distinct isotopic pattern benefitted from the high resolution of the Orbitrap MS analyzer. (C) MS/MS spectra using collision-induced dissociation (CID) fragmentation of A2M (444.78 m/z) precursor peptide, colored peaks (red for b ions and blue for y ions) indicate matches between experimental and theoretical/calculated values.

Figure 3

Distribution of the mean values (A) and coefficient of variation (B) of proteins detected in the human aqueous humor samples. The proteins were subdivided into four categories, based on their detection rate. High: detected in >75%; Medium: detected in 50–75%; Low: detected in 25–50%; Rare: detected in <25% of the samples. Coefficient of variation decreases as mean protein expression increases.

Figure 4

Biological processes (A), cellular components (B), and molecular functions (C) associated with the highly abundant aqueous humor proteins (detected in >50% samples). Bioinformatics analysis was performed in order to associate significantly enriched Gene Ontology (GO) terms to the constitutive aqueous humor proteome. The horizontal bars represent the number of proteins annotated to each GO term, and the black lines represent the p-value of enrichment.

Figure 5

Three top-scoring interaction networks of highly abundant aqueous humor proteins. Ingenuity Pathway Analysis (IPA) was performed on the 243 proteins detected in at least half of the aqueous humor samples. (A) Network 1: includes several members of the Apolipoprotein, Complement, and SERPIN families. (B) Network 2: Network of proteins involved in tissue development, protein synthesis, and cellular compromise. (C) Network 3: Protein cluster associated with humoral immune response, inflammatory response, and protein synthesis. Each protein is represented as a node, and edges represent interactions between proteins. The intensity of color represents the relative levels of proteins (brighter red nodes indicate higher levels). Proteins are separated based on the cellular compartments.

Figure 6

Race-specific differences in human aqueous humor proteins. A total of six proteins were upregulated (A) and five proteins were downregulated (B) in African American subjects as compared to Caucasian subjects.