Proteomic analyses of corneal tissue subjected to alkali exposure.
OBJECTIVE
To determine whether exposure to alkaline chemicals results in predictable changes in corneal protein profile. To determine whether protein profile changes are indicative of severity and duration of alkali exposure.


METHODS
Enucleated bovine and porcine (n = 59 each) eyes were used for exposure to sodium, ammonium, and calcium hydroxide, respectively. Eyes were subjected to fluorescein staining, 5-bromo-2'-deoxy-uridine (BrdU) labeling. Excised cornea was subjected to protein extraction, spectrophotometric determination of protein amount, dynamic light scattering and SDS-PAGE profiling, mass spectrometric protein identification, and iTRAQ-labeled quantification. Select identified proteins were subjected to Western blot and immunohistochemical analyses.


RESULTS
Alkali exposure resulted in lower protein extractability from corneal tissue. Elevated aggregate formation was found with strong alkali exposure (sodium hydroxide>ammonium, calcium hydroxide), even with a short duration of exposure compared with controls. The protein yield after exposure varied as a function of postexposure time. Protein profiles changed because of alkali exposure. Concentration and strength of the alkali affected the profile change significantly. Mass spectrometry identified 15 proteins from different bands with relative quantification. Plexin D1 was identified for the first time in the cornea at a protein level that was further confirmed by Western blot and immunohistochemical analyses.


CONCLUSIONS
Exposure to alkaline chemicals results in predictable and reproducible changes in corneal protein profile. Stronger alkali, longer durations, or both, of exposure resulted in lower yields and significant protein profile changes compared with controls.
Parikh
Toral
T
Bascom Palmer Eye Institute, University of Miami, Miami, Florida, USA.
Eisner
Natalie
N
Venugopalan
Praseeda
P
Yang
Qin
Q
Lam
Byron L
BL
Bhattacharya
Sanjoy K
SK
D064753:Ammonium Hydroxide; D000818:Animals; D015153:Blotting, Western; D002126:Calcium Hydroxide / Q000633:toxicity*; D002417:Cattle; D003315:Cornea / Q000187:drug effects* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D015353:Eye Enucleation; D005136:Eye Proteins / Q000378:metabolism*; D019793:Fluorescein / Q000378:metabolism; D015590:Fluorophotometry; D006878:Hydroxides / Q000633:toxicity*; D020287:In Situ Nick-End Labeling; D013058:Mass Spectrometry; D040901:Proteomics; D012972:Sodium Hydroxide / Q000633:toxicity*; D013552:Swine
D005136:Eye Proteins; D006878:Hydroxides; D064753:Ammonium Hydroxide; D012972:Sodium Hydroxide; D002126:Calcium Hydroxide; D019793:Fluorescein