Abstract
Purpose :
The lens sutures represent the major influx pathway of the lens microcirculation system, yet little is known about the protein distribution within the sutures and how this changes with fiber cell age. The purpose of this work is to characterize the lens suture proteome and define changes in protein expression with location, i.e. fiber cell age.
Methods :
Imaging mass spectrometry (IMS) and spatially-resolved proteomics methods were applied to characterize the lens suture proteome at various depths from both anterior and posterior poles of bovine lenses. Frozen and fixed bovine lenses were sectioned and processed for either matrix-assisted laser desorption ionization (MALDI) IMS or for laser capture microdissection of suture and adjacent/non-suture regions. For IMS studies, sections were washed to remove soluble proteins and trypsin was sprayed onto the tissue sections to generate peptides from all proteins present. For LCM-proteomics analysis, captured tissue was subjected to trypsin digestion and either data-dependent acquisition (DDA) or data-independent acquisition (DIA) LC-MS/MS analysis. Validation of observed protein expression differences was accomplished by immunohistochemical (IHC) analysis of targeted proteins.
Results :
IMS revealed highly abundant signals for ARVCF and N-cadherin in the Y-shaped sutures and connexins 50 and 46 in non-suture regions in both anterior and posterior poles. These localizations were confirmed by IHC analysis. Quantitative proteomics analysis confirmed the ARVCF and N-cadherin results and provided a rich database of ~700 suture-related proteins. Interestingly, GLUT1 and GLUT3 were enriched in suture regions and more abundant in the inner cortex suture region and in the posterior suture compared to the outer cortex and anterior suture, respectively. Adherens junction proteins were more abundant in the outer cortex suture where there is greater extracellular space between suture tips; this trend was not observed in the inner cortex suture.
Conclusions :
Key suture proteins have been identified via spatially-resolved proteomics methodologies. Differential GLUT1 and GLUT 3 localization may play an important role in delivering glucose and dehydroascorbic acid to fiber cells of the inner cortex and posterior poles.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.