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Jamie L. Wenke, David B. Friedman, Kevin L. Schey; Development of a Quantitative, High Resolution Spatial Profiling Method for AQP0 Modifications. Invest. Ophthalmol. Vis. Sci. 2012;53(14):1044.
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To optimize sample preparation and experimental conditions to quantitatively map the spatial distribution of AQP0 modifications in the human lens with high spatial resolution and little interference from soluble proteins.
MALDI Imaging: human lenses were removed from whole eyes and frozen immediately. Twenty micron equatorial sections were placed on a MALDI target using MeOH soft landing and washed extensively with water. Several solvents were investigated for their ability to remove undesirable soluble proteins. Sinapinic acid was spotted on the tissue at 200 µm resolution using a Labcyte Portrait 630. Spectra were acquired on a Bruker Autoflex Speed, and ion images were generated using Bruker FlexImaging software. On-tissue tryptic digestion of water and urea washed sections was employed followed by sinapinic acid matrix spotting and tandem TOF/TOF analysis to confirm protein assignments.Laser Capture Microdissection Targeted LC-MS/MS Analysis: fresh human lenses were fixed (75:25 EtOH:AcOH) and paraffin-embedded. Sections were placed on Zeiss PEN membrane slides and deparaffinized prior to staining. A Zeiss instrument was used for precise morphology-directed capture of tissue from specific regions of the cortex and nucleus. Sections from each region were pooled (~106 µm2 tissue per region), membranes prepared and digested with Endoproteinase Lys-C. Samples were analyzed by LC-MS/MS, and AQP0 levels were quantified by multiple reaction monitoring using internal isotopically-labeled peptide standards.
Three 1% formic acid washes, with water washes in between, were found to be essential for removing crystallins and improving AQP0 signal. With this wash, less ACN was required for AQP0 co-crystallization with the matrix. Phosphorylated AQP0 was mass resolved from unmodified AQP0 and pAQP0 was localized in a distinct cortical region of the human lens. LCM combined with targeted quantitative LC-MS/MS analysis allowed AQP0 modifications to be correlated to membrane morphology changes.
These methods confirm that AQP0 is phosphorylated most abundantly in a well-defined region of the lens cortex. LCM combined with targeted quantitative proteomics provides sensitive, high spatial resolution, and quantitative information that can be correlated with cell membrane morphology.
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