Proteome analysis of human aqueous humor

Abstract

Purpose: Human aqueous humor (hAH) provides nutrition and immunity within the anterior chamber of the eye. Characterization of the protein composition of hAH will identify molecules involved in maintaining a homeostatic environment for anterior segment tissues. The present study was conducted to analyze the proteome of hAH.

Methods: hAH samples obtained during elective cataract surgery were divided into three matched groups and immunodepleted of albumin, IgG, IgA, haploglobin, antitrypsin, and transferrin. Reduced and denatured proteins (20 μg) from each group were separated by gel electrophoresis. Thirty-three gel slices were excised from each of three gel lanes (n = 99), digested with trypsin, and subjected to nanoflow liquid chromatography electrospray ionization tandem mass spectrometry (nano-LC-ESI-MS/MS). The protein component of hAH was also analyzed by antibody-based protein arrays, and selected proteins were quantified.

Results: A total of 676 proteins were identified in hAH. Of the 355 proteins identified by nano-LC-ESI-MS/MS, 206 were found in all three groups. Most of the proteins identified by nano-LC-ESI-MS/MS had catalytic, enzymatic, and structural properties. Using antibody-based protein arrays, 328 cytokines, chemokines, and receptors were identified. Most of the quantified proteins had concentrations that ranged between 0.1 and 2.5 ng/mL. Ten proteins were identified by both nano-LC-ESI-MS/MS and antibody protein arrays.

Conclusions: Proteomic analysis of hAH identified 676 nonredundant proteins. More than 80% of these proteins are novel identifications. The elucidation of the aqueous proteome will establish a foundation for protein function analysis and identification of differentially expressed markers associated with diseases of the anterior segment.

Figure 1.

Immunodepletion of hAH. (A) Eighty-five hAH samples were divided into three groups and immunodepleted of albumin, transferrin, antitrypsin, haploglobin, IgG, and IgA. Groups were separated on 10% to 14.5% SDS-PAGE gradient gels. PD, predepletion; D, immunodepleted (flow-thru); E, eluted from column. (B) Thirty-three gel slices were isolated from each group, independently trypsinized, and processed for nano-LC-ESI-MS/MS. G1, group 1; G2, group 2: G3, group 3.

Figure 2.

The number of hAH proteins identified by nano-LC-ESI-MS/MS. (A) The Venn diagram showing proteins that are common and unique between groups 1, 2, and 3. (B) Distribution by function of all 355 proteins identified by nano-LC-ESI-MS/MS.

Figure 3.

Verification of BigH3, fibulin-3, and myocilin in hAH. Three proteins—BigH3, fibulin-3, and myocilin—were assessed by Western blot for their presence in hAH. All three proteins were determined to be present in the three groups by nano-LC-ESI-MS/MS and were confirmed by Western blot analysis.

Figure 4.

Presence and absence of several TGFβ family members in hAH. Representative images of several TGFβ-family members and their corresponding fluorescent signal intensity from antibody-based protein arrays. TGFβ2, TGFβ3, type I, and type II receptors were present in all four groups (groups 4–7). TGFβ1 was present in groups 4 and 5, but was “absent” in groups 6 and 7 because of low signal strength of less than threefold above background. TGFβ5 was negative in all groups.