Identification Of Phosphorylation Sites In Human Age Related Cataract Lens

Cataract is the main cause of vision impairment in the world, and age-related cataract (ARC) is the most common reason of blindness in elder people. To date, surgery is still the only way to cure cataract, but the high cost has become a tremendous burden for our society. Therefore, preventing or delaying ARC is of great significance.Protein phosphorylation, one of the most important protein post translational modifications, plays a key role in regulating many cellular processes such as proliferation, differentiation, and signal transduction. However, the abnormality of this reversible phosphorylation in proteins may result in a wide variety of diseases. As the major protein components of the vertebrate eye lens, crystallins have to last the life time and retain lens transparence. For the aforementioned reasons, we believe that the imbalanced protein phosphorylation of crystallins may result in the early change of lens before the irreversible crystallin denaturation. Therefore, preventing cataract before the irreversible protein denaturation is considered to be the optimum option. In addition, identification of phosphorylation sites in crystallins is the first step of functional study.In this study, we used in situ tryptic digestion combined with matrix-assisted laser desorption/ionization (MALDI) mass spectrometry to identify and compare the phosphorylation sites in crystallins of a human transparent lens and an age related cataract lens. In the transparent lens tissue, four crystallins were identified to contain a total of eight sites of phosphorylation. While in the ARC lens, six crystallins were found to contain eleven sites of phosphorylation, nine of which were previously unreported.In summary, a method for in situ tryptic digestion combined with MALDI tissue identification of crystallin phosphorylation products has been developed. The observations of phosphorylation sites will be useful in spatial distribution imaging and functional study. Methods:(1) Frozen human lenses were equatorially and axially cryosectioned into10μm to20μm thick sections under the temperature of-10℃to-25℃respectively, and the sections were mounted onto glass slides. Dissecting microscope was used to determine the optimum temperature and thickness of lens cryosection.(2) Cytochrome C and myoglobin were used as standard proteins to optimize the experimental condition of MALDI mass spectrometry.Results:The optimum cryosection temperature was-18℃and the section thickness is14μm. The optimal matrix was composed of2.5mg/mL alpha-Cyano-4-hydroxycinnamic acid (CHCA) with0.1%trifluoracetic acid (TFA) and0.6-0.8mg/ml citric acid ammonia. Two layers of matrix were sprayed on the section with the velocity of200mm/min at the temperature of120℃. MALDI mass spectra were acquired in reflection mode between m/z700and4000with a Nd:YAG laser at355nm, a repetition rate of200Hz, and an acceleration voltage of20kV. Two thousand laser shots per spot were accumulated at50different spot positions. The data was further analyzed by Data Explorer4.5.Conclusions:We optimized the experimental condition of lens cryosection and MALDI mass spectrometry and laid the foundation for further study. Purpose:The purpose of this part was to identify and compare the phosphorylation sites in crystallins of a human transparent lens and an age related cataract lens using in situ tryptic digestion combined with matrix-assisted laser desorption/ionization mass spectrometry.Methods:(1) Frozen human lenses were equatorially and axially cryosectioned into14-μm-thick sections at the temperature of-18℃, and the sections were mounted onto glass slides.(2) Myoglobin, bovine serum albumin (BSA) and a-casein were used as standard proteins to test the efficiency of Graphene-Immobilized Trypsin Reactor.(3) Proteins in the lens sections were digested with Graphene-Immobilized Trypsin Reactor and subjected to MALDI mass spectrometric analysis for identification of modifications. The data was further analyzed by Data Explorer4.5.Results:In the transparent lens tissue, four crystallins were identified to contain a total of eight sites of phosphorylation. While in the ARC lens, six crystallins were found to contain eleven sites of phosphorylation, nine of which were previously unreported.Conclusions:In summary, a method for in situ tryptic digestion combined with MALDI tissue identification of crystallin products has been developed. The observations of phosphorylation sites will be useful in spatial distribution imaging and functional study.