Purpose :
To better understand the influence of the aqueous humor proteome on ocular physiology, it is critical to differentiate between aqueous proteins derived from ocular structures from blood-derived proteins that diffuse into aqueous humor. By comparing the concentration of various proteins in aqueous humor and in plasma, we sought to identify proteins that are present in higher concentrations than would be expected from blood-derived proteins. A similar method has been used for the cerebrospinal fluid (CSF) proteome..

Methods :
Aqueous humor and blood samples were obtained during cataract surgery on an IRB-approved protocol. For high abundance proteins, liquid chromatography tandem mass spectrometry (LC-MS/MS) was used to screen for proteins that could be quantified by ELISA. ELISA was performed on 40 aqueous humor and 26 plasma samples. For low abundance proteins, 174 proteins were screened by protein-antibody (Quantibody) arrays for their presence in aqueous humor. Of these, 70 were within the linear range of the standards and were further analyzed using a set of 104 aqueous and 20 plasma samples. The aqueous-plasma ratio (AQ:PL) was calculated for each protein.

Results :
A total of 110 proteins were found that had reliable concentration measurements in both aqueous humor and plasma. By LC-MS/MS, we identified 56 high confidence proteins, of which 14 were unique to aqueous humor and 42 were plasma proteins. ELISA data was obtained on 40 proteins while Quantibody array data was obtained on 70 proteins. Various AQ:PLs included 11:1 for osteopontin (MW 44 kDa), 1:304 for albumin (MW 67 kDa), 1:576 for IgG (MW 150 kDa), and 1:3467 for alpha-2 macroglobulin (MW 718 kDa). The correlation between log(AQ:PL) and molecular weight (MW) was -0.58 (p=0.003) and the correlation between log(AQ:PL) and log(MW) was -0.74 (p<0.0001).

Conclusions :
Protein concentration in aqueous humor, relative to plasma, is non-linearly dependent on MW. Similar to CSF, the concentration of blood-derived proteins in aqueous humor appears to be the consequence of passive, molecular size-dependent diffusion, although the AQ:PLs are slightly lower than published CSF:PLs. A small fraction of aqueous humor proteins originate within the eye and for some of these proteins, the blood contributes a non-negligible fraction to the aqueous concentration. The AQ:PL of similarly sized proteins may help predict the blood-derived contribution for these proteins.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.