Proteome analysis of human aqueous humor.
OBJECTIVE
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.
Chowdhury
Uttio Roy
UR
Department of Ophthalmology, Mayo Clinic, Rochester, Minnesota, USA.
Madden
Benjamin J
BJ
Charlesworth
Mary Christine
MC
Fautsch
Michael P
MP
D000368:Aged; D000369:Aged, 80 and over; D001082:Aqueous Humor / Q000737:chemistry*; D015153:Blotting, Western; D002853:Chromatography, Liquid; D016207:Cytokines / Q000032:analysis; D004797:Enzyme-Linked Immunosorbent Assay; D016326:Extracellular Matrix Proteins / Q000032:analysis; D005136:Eye Proteins / Q000032:analysis*; D005260:Female; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000032:analysis; D008297:Male; D008875:Middle Aged; D020543:Proteome / Q000032:analysis*; D040901:Proteomics; D021241:Spectrometry, Mass, Electrospray Ionization; D053719:Tandem Mass Spectrometry; D016212:Transforming Growth Factor beta / Q000032:analysis; D000099222:betaIG-H3 Protein
D016207:Cytokines; D016326:Extracellular Matrix Proteins; D005136:Eye Proteins; D036341:Intercellular Signaling Peptides and Proteins; D020543:Proteome; D016212:Transforming Growth Factor beta; D000099222:betaIG-H3 Protein; C119630:EFEMP1 protein, human
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