Proteomics analysis of mouse lens supernatant treated with hydrogen peroxide in vitro

Objective

To study the protein leakage of mouse lens treated with hydrogen peroxide in vitro, and to identify and carry out bioinformatics analysis of the leaked proteins.

Methods

Intact binocular lenses of 42 healthy male C57BL/6J mice were enucleated following sacrifice, and 28 lenses were arbitrarily combined as one sample, with 3 samples in total.Mouse lenses were cultured with medium containing 2 mmol/L hydrogen peroxide for 24 hours to establish a cataract model in vitro.Blank medium M-199 was taken as control sample.Supernatant was subjected to polyacrylamide gel electrophoresis (PAGE). The structure of the supernatant proteins was identified by liquid chromatography coupled with tandem mass spectrometry, and the high-abundant proteins were quantitatively analyzed by label-free quantitative mass spectrometry.The biological information of the identified proteins was analyzed by METASCAPE database.Content of cytokines in the supernatant was determined using the multiplex bead immunoassay system.This study protocol was approved by an Ethics Committee of The First Affiliated Hospital of Chongqing Medical University (No.2019-173), and the use and care of experimental animals complied with the ARVO statement.

Results

After 24-hour culturing, the lenses became turbid, and the capsules were intact.The protein concentration in the supernatant was (3.73±0.59)μg/μl.PAGE analysis of the supernatant protein profile revealed that protein bands were below 35 000.A total of 675 proteins were identified with mass spectrometry.Sixteen crystallin proteins accounted for (86.1±0.8)% of the total protein abundance; the biological processes those proteins participated in mainly included cell-cell adhesion, cell transformation, oxidation-reduction, inhibition of apoptosis, and protein translocation; the molecular functions of those proteins mainly involved the activity of hydrolase, oxidoreductase, catalysis, translation initiation factor, and GTPase; the subcellular localization of proteins were mainly in cytoplasm, extracellular exosome, the nucleus, the plasma membrane, and the intercellular space.The results of an antibody-based multiplex bead immunoassay presented 9 cytokines in the supernatant, which included monocyte chemotactic protein-1 and transforming growth factor-β₂ highly expressed in intraocular fluid.

Conclusions

There are protein leakages in lens treated with hydrogen peroxide in vitro, and it may have influences on metabolism and/or functions of other intraocular tissues.

Cataract; Lens; Supernatant; Proteomics; Cytokines