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Spatially Directed Proteomics of the Human Lens Outer Cortex Reveals an Intermediate Filament Switch Associated With the Remodeling Zone.

Abstract：

PURPOSE:To quantify protein changes in the morphologically distinct remodeling zone (RZ) and adjacent regions of the human lens outer cortex using spatially directed quantitative proteomics. METHODS:Lightly fixed human lens sections were deparaffinized and membranes labeled with fluorescent wheat germ agglutinin (WGA-TRITC). Morphology directed laser capture microdissection (LCM) was used to isolate tissue from four distinct regions of human lens outer cortex: differentiating zone (DF), RZ, transition zone (TZ), and inner cortex (IC). Liquid chromatography-tandem mass spectrometry (LC-MS/MS) of the plasma membrane fraction from three lenses (21-, 22-, and 27-year) revealed changes in major cytoskeletal proteins including vimentin, filensin, and phakinin. Peptides from proteins of interest were quantified using multiple reaction monitoring (MRM) mass spectrometry and isotopically-labeled internal peptide standards. RESULTS:Results revealed an intermediate filament switch from vimentin to beaded filament proteins filensin and phakinin that occurred at the RZ. Several other cytoskeletal proteins showed significant changes between regions, while most crystallins remained unchanged. Targeted proteomics provided accurate, absolute quantification of these proteins and confirmed vimentin, periplakin, and periaxin decrease from the DF to the IC, while filensin, phakinin, and brain acid soluble protein 1 (BASP1) increase significantly at the RZ. CONCLUSIONS:Mass spectrometry-compatible fixation and morphology directed laser capture enabled proteomic analysis of narrow regions in the human lens outer cortex. Results reveal dramatic cytoskeletal protein changes associated with the RZ, suggesting that one role of these proteins is in membrane deformation and/or the establishment of ball and socket joints in the human RZ.

journal_name

Invest Ophthalmol Vis Sci

journal_title

Investigative ophthalmology & visual science

authors

Wenke JL,McDonald WH,Schey KL

doi

10.1167/iovs.16-19791

subject

Has Abstract

pub_date

2016-08-01 00:00:00

pages

4108-14

issue

eissn

0146-0404

issn

1552-5783

pii

2545649

journal_volume

pub_type

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Spatially Directed Proteomics of the Human Lens Outer Cortex Reveals an Intermediate Filament Switch Associated With the Remodeling Zone.