Strategies to recover proteins from ocular tissues for proteomics

Nikhil Patel, Ekta Solanki, Renata Picciani, Valerie Cavett, Jennifer A. Caldwell-Busby, Sanjoy K. Bhattacharya

Research output: Contribution to journalArticle

45 Scopus citations

Abstract

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 μg 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.

Original languageEnglish (US)
Pages (from-to)1055-1070
Number of pages16
JournalProteomics
Volume8
Issue number5
DOIs
StatePublished - Mar 1 2008

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Keywords

  • Electrospray ionization-tandem mass spectrometry
  • Protein solubilization
  • Proteome profiling
  • Shotgun proteomics

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

Cite this

Patel, N., Solanki, E., Picciani, R., Cavett, V., Caldwell-Busby, J. A., & Bhattacharya, S. K. (2008). Strategies to recover proteins from ocular tissues for proteomics. Proteomics, 8(5), 1055-1070. https://doi.org/10.1002/pmic.200700856