Strategies to recover proteins from ocular tissues for proteomics.
We present here the results of protein extraction from different ocular regions using different detergents. Extraction strategies used to determine optimal protein extraction included: pressure cycling and aqueous-organic phase extraction in combination with electrophoretic fractionation for anterior, posterior, and peripapillary sclera. Detergent extraction of proteins from freshly enucleated porcine eyes (n = 8) showed significant differences for different eye regions. Protein yield ranged from 2.3 to 50.7 mug protein/mg for different ocular tissues, with the lens yielding the most protein. ASB-14 and Triton X-100 provided the best protein yields (n = 10) for anterior and posterior sclera. The spectrophotometric measurements for ASB-14 were not consistent with SDS-PAGE densitometry. A combination of 0.5% Triton X-100, 0.5% Tween-20, and 0.1% Genapol C-100 was found optimal for extraction from sclera. Proteins from different regions of the eye are best extracted with different detergents. The pressure cycling technology provided superior extraction compared to the other methods. Additional aqueous-organic phase partitioning enables superior fractionation when compared to SDS-PAGE alone. Organic phase fractionation is compatible with MS and allowed identification of 34, 71, and 77 proteins respectively from anterior, posterior, and peripapillary sclera. The extraction strategy may affect the final outcome in protein profiling by MS or by other methods.
Patel
Nikhil
N
Bascom Palmer Eye Institute, University of Miami, Miami, FL, USA.
Solanki
Ekta
E
Picciani
Renata
R
Cavett
Valerie
V
Caldwell-Busby
Jennifer A
JA
Bhattacharya
Sanjoy K
SK
D000818:Animals; D003902:Detergents; D005123:Eye / Q000737:chemistry*; D005136:Eye Proteins / Q000032:analysis*; D006801:Humans; D013058:Mass Spectrometry; D040901:Proteomics; D012590:Sclera / Q000737:chemistry; D013552:Swine
D003902:Detergents; D005136:Eye Proteins