Abstract
Purpose: :
To characterize the tissue distributions, with high spatial resolution, of modifications to both lens soluble and integral membrane proteins.
Methods: :
Laser capture microdissection (LCM)/mass spectrometry (MS) and direct tissue profiling/imaging MS methods were used to analyze protein modifications from lens tissue sections. Lens cryosections were subjected to LCM to extract lens regions of interest. Captured fiber cells were prepared and analyzed by HPLC/MS/MS to determine membrane protein content and modifications. Direct tissue analysis of lens membrane proteins was accomplished by, first, washing the cryosections, followed by MALDI-MS profiling of discrete tissue locations where MALDI matrix was applied. MALDI-MS images of lens soluble proteins was accomplished by spray coating the surface of the cryosection with MALDI matrix followed by rastering the MALDI laser over the entire surface of the tissue.
Results: :
LCM-MS/MS results indicate extensive sequence coverage of integral membrane proteins: AQP0, MP20, connexins 46 & 50, as well as AQP5. Major sites of phosphorylation were identified in these proteins and spatial differences in the extent of modification were observed. Direct tissue profiling revealed age-related truncation of AQP0 as well as a novel AQP0 modification. MALDI images of soluble lens proteins revealed large numbers of modified crystallin forms and their spatial distributions. A ring of alpha-crystallin phosphorylation was observed specific to the inner cortical region in all lenses examined.
Conclusions: :
A combination of spatially-resolved proteomics approaches provides information on the distribution of modified lens proteins in both soluble and membrane fractions. Truncation and phosphorylated forms are located in specific regions of the lens indicating age- or developmentally-related changes in protein structures.
Keywords: protein modifications-post translational • proteomics