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K.L. Schey, J. Han; Laser Capture Microdissection–Proteomics Analysis of Changes in the Lens Membrane Proteome During Fiber Cell Maturation . Invest. Ophthalmol. Vis. Sci. 2005;46(13):1867.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: to examine changes in the lens membrane proteome during fiber cell maturation. Methods: Laser capture microdissection (LCM) from 12–14 µm thick bovine and human lens sections was used to capture lens tissue containing fiber cells of different ages and corresponding to specific lens regions. After buffer washes and stripping of membranes by urea and NaOH washes, membrane proteins were subjected to tryptic digestion. Both MALDI tandem mass spectrometry and HPLC–MS/MS were used to identify changes in membrane protein expression and modification. Data analysis by Mascot and Sequest were carried out for protein identification. Results: Approximately 2 min. LCM capture time was required for successful MALDI–MS/MS analysis and 1 hour capture time for LC–MS/MS analysis. Over 30 proteins were identified in a single LCM–MS experiment by LC–MS/MS. Changes in protein and protein modification between equatorial and nuclear regions include oxidation, phosphorylation and truncation on membrane or membrane–associated proteins, especially on the lens integral membrane protein ––– aquaporin 0. Conclusions: LCM–mass spectrometry analysis provides a powerful approach to examine changes in lens membrane proteome during fiber cell maturation. This approach will generate new information on the regulation of fiber cell differentiation and maintenance of fiber cell homeostasis via the membrane proteome.
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