Abstract
Purpose :
The lacrimal gland (LG) is the major source of aqueous tears, and insufficient LG secretion leads to dry eye disease (DED).To provide a foundational description of LG's protein expression patterns, we have prepared protein extracts of LGs from wild-type and DED model mice and analyzed the proteome by quantitative mass spectrometry (MS).
Methods :
Lacrimal glands (LG) were isolated from a DED mouse model, male NOD (non-obese diabetic) and control wild-type BALB/c mice (n=6 each). Protein samples were prepared in 8 M urea-based lysis buffer and protein concentrations determined by the BCA method. The equivalent of 200 μg protein were tryptically digested and analyzed by nanoflow liquid chromatography tandem mass spectrometry (LC-MS/MS). Proteins were identified and quantified using the PEAKS X bioinformatics suite. Downstream differential protein expression analysis was performed using the MS-DAP R package. Selected significantly differentially expressed proteins were subjected to spatial expression analysis using immunohistochemistry.
Results :
Cumulatively, the LC-MS/MS-based proteomics analyses of the murine LG samples identified a total of 31,932 peptide sequences resulting in 2617 protein identifications at a 1% false discovery rate at the peptide and protein level. PCA and hierarchical cluster analysis revealed a separation of NOD and BALB/c samples. Further, we observed that the overall protein diversity was consistently higher in NOD samples. After applying global peptide filter criteria and peptide-to-protein rollup, 1981 remaining proteins were subjected to differential expression analysis using the MSqRob algorithm which identified 531 statistically significant protein abundance changes. Furthermore, using a differentially detection approach, we identified 112 and 17 proteins that were exclusively expressed in NOD and BALB/c samples, respectively. At the cellular level, the up-regulated expression of clusterin and down-regulated expression of arginase-1 were confirmed by immunohistochemistry.
Conclusions :
Our data suggest that chronic inflammation leads to significant alterations in the lacrimal gland proteome. Ongoing studies aim to identify potentially unique, inflammation-induced peptides/proteins that could be amenable to pharmacological modulation.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.