MALDI Imaging Mass Spectrometry of Integral Membrane Proteins from Ocular Lens and Retinal Tissue†

Angus C. Grey, Pierre Chaurand, Richard M. Caprioli and Kevin L. Schey*
Mass Spectrometry Research Center and Department of Biochemistry, Vanderbilt University Medical Center, Nashville, Tennessee
J. Proteome Res., 2009, 8 (7), pp 3278–3283
DOI: 10.1021/pr800956y
Publication Date (Web): March 30, 2009
Copyright © 2009 American Chemical Society
* To whom correspondence should be addressed. Kevin L. Schey, Mass Spectrometry Research Center, Vanderbilt University Medical Center, Suite 9160, Medical Research Building III, 465 21st Ave. So, Nashville, TN 37232-8575. Phone: 615-936-6861. Fax: 615-343-8372. E-mail: kevin.schey@vanderbilt.edu., †

Originally submitted and accepted as part of the “Tissue Proteomics and Metabolomics” special section, published in the April 2009 issue of J. Proteome Res. (Vol. 8, No. 4).

Biochemical Methods

Abstract

Abstract Image

A tissue preparation protocol for MALDI (matrix-assisted laser desorption/ionization) imaging mass spectrometry of integral membrane proteins was developed using ocular lens and retinal tissues as model samples. Frozen bovine and human lenses were cryosectioned equatorially or axially at −20 °C into 20 μm-thick tissue sections. Lens sections were mounted onto gold-coated MALDI targets by methanol soft-landing to maintain tissue integrity. Tissue sections underwent extensive water washing to deplete the samples of highly abundant water-soluble proteins. Automated matrix deposition was achieved using an acoustic reagent multispotter, with sinapinic acid as matrix and high percentage acetonitrile as solvent, with a center-to-center spot spacing of 200−300 μm. Molecular images of full-length Aquaporin-0 (AQP0) and its most abundant truncation products were obtained from mass spectral data acquired across whole bovine and human lens sections. In equatorial and axial sections of bovine lenses, full-length AQP0 was detected throughout the lens. A truncation product corresponding to AQP0 (1−260) was detected in the bovine lens core at low abundance. In axial lens sections, no antero-posterior variation was detected. In 11 year-old human lens sections, full-length AQP0 was most abundant in the lens periphery, but was detected throughout the lens. The major truncation product, consisting of AQP0 residues 1−246, was absent from the lens periphery and increased in abundance in the lens core. This tissue preparation protocol was then applied to image the distribution of the G-protein coupled receptor, opsin, in the rabbit retina. This protocol has expanded the variety of target analytes which can be detected by MALDI imaging mass spectrometry to include intact integral membrane proteins.

Keywords:

MALDI imaging mass spectrometry; AQP0; membrane protein; lens
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History

  • Published In Issue July 06, 2009
  • Article ASAPJune 11, 2009
  • Just Accepted ManuscriptMarch 30, 2009
  • Received: November 4, 2008

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