Quantitative Analysis of Lens Membrane Protein Modifications with Fiber Cell Morphology

Abstract

Purpose: To quantify phosphorylated, fatty acid acylated, and unmodified aquaporin-0 (AQP0) peptides in human lens regions based on fiber cell age and cell membrane morphology.

Methods: Human lenses were lightly fixed, paraffin-embedded, and sectioned equatorially at 10µm. Sections were stained with Oil Red O to visualize plasma membranes. Laser capture microdissection (LCM) was used to capture small areas of cells (10³ µm²) with distinct morphology at specific distances from the lens capsule. Tissue was captured and pooled (10⁵ µm² total area), then washed to prepare membrane fractions and digested with Lys-C. Four regions representing unique fiber cell ages were collected: differentiating zone (DZ), remodeling zone (RZ), transition zone (TZ), and outer nucleus (ON). Young (0-35), middle-aged (36-64), and older (65+) lenses were analyzed. Isotopically-labeled peptides from AQP0, corresponding to phosphorylated and unmodified ₂₂₉SISERL(p)SVLK₂₃₈, were synthesized by Sigma. Characteristic fragmentation transitions for each peptide were selected. Labeled peptides were spiked into each fraction as internal standards to quantify endogenous peptide levels. Samples were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS), using multiple reaction monitoring for quantitation.

Results: Fixation and sectioning were optimized to leave the lens capsule and outer cell layers intact; this analysis is not possible with standard cryosectioning. A quantitative difference in phosphorylated, fatty acid acylated, and unmodified AQP0 peptide levels was detected between different regions of the lens, similar to previous reports that employed lower spatial resolution methods. The changes in AQP0 phosphorylation and fatty acid modification were mapped to regions of distinct cell membrane morphology, which is correlated with fiber cell age. Our high spatial resolution measurements are the first to demonstrate protein changes directly related to the reported membrane remodeling that occurs in outer regions of the lens.

Conclusions: The combination of LCM and quantitative proteomics is a powerful method for studying protein modifications based on morphological features of lens fiber cells. Our results shed light on AQP0 modifications correlated with fiber cell age and cell membrane morphology, and support our understanding of AQP0 changes in structure and function that coincide with lens membrane remodeling.

Keywords: 657 protein modifications-post translational