May 2007
Volume 48, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2007
Spatial Distribution of AQP0 Modifications Using MALDI Tissue Profiling
Author Affiliations & Notes
  • D. Thibault
    Department of Cell and Molecular Pharmacology and Experimental Therapeutics, Medical University of South Carolina, Charleston, South Carolina
  • C. Gillam
    Wofford College, Spartanburg, South Carolina
  • K. Schey
    Department of Cell and Molecular Pharmacology and Experimental Therapeutics, Medical University of South Carolina, Charleston, South Carolina
  • Footnotes
    Commercial Relationships D. Thibault, None; C. Gillam, None; K. Schey, None.
  • Footnotes
    Support NIH Grant EY13462
Investigative Ophthalmology & Visual Science May 2007, Vol.48, 4212. doi:
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      D. Thibault, C. Gillam, K. Schey; Spatial Distribution of AQP0 Modifications Using MALDI Tissue Profiling. Invest. Ophthalmol. Vis. Sci. 2007;48(13):4212.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose:: To develop and apply a method for the direct tissue profiling of membrane proteins by MALDI-MS to examine the spatial distribution of AQP0 modifications throughout the lens.

Methods:: Frozen, mature bovine lenses and various aged human lenses were decapsulated and sectioned equatorially at a temperature of -20 to -21 °C to a thickness of 20 µm, using a HM505E Microm. Sections from the equator region of the lens were transferred to a MALDI plate covered with a thin layer of anhydrous ethanol. Three regions of the lens sections were profiled: outer cortex, inner cortex, and nucleus. Within each region, areas were washed with water and then spotted with 0.5 µl of 7:3 formic acid:hexafluoroisopropanol. While still wet, the area was spotted with either saturated 3,5-dimethoxy-4-hydroxycinnamic acid matrix (sinapinic acid, SA) in 90% MeCN, 0.1% TFA or saturated 2,5-dihydroxybenzoic acid (DHB) matrix in 80% MeCN, 0.1% TFA, 0.3 mg/ml ammonium citrate. MALDI mass spectra were acquired in spotted areas using an Applied Biosystems Voyager-STR instrument. Membrane preparations were also generated from dissected lens regions to validate the direct tissue profiling results.

Results:: The two most abundant membrane proteins of the lens, AQP0 and MP20, were detected directly from lens sections by MALDI tissue profiling. The spatial distribution of AQP0 throughout the lens suggests that truncation products increase with fiber cell age in both bovine and human lenses; this is in agreement with previous reports. Importantly, a novel modification to AQP0 in both bovine and human lenses was detected using this method, and this modified AQP0 localized specifically to the inner cortex.

Conclusions:: A MALDI tissue profiling method was developed for the analysis of the spatial distribution of integral membrane proteins directly from tissue sections. The distribution of AQP0 obtained with this method indicates increased truncation in aged fiber cells and a novel modification that is preferentially localized to inner cortical fiber cells in bovine and human lenses.

Keywords: protein modifications-post translational • aging 
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