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Title: Proteomics in lens induced myopic (LIM) mammalian model
Authors: Bian, Jingfang
Degree: Ph.D.
Issue Date: 2021
Abstract: Myopia is a common refractive error. The prevalence of myopia is approximately 30% worldwide and is predicted to reach 50% by 2050. Yet, molecular mechanisms of myopia remain obscure. Traditional myopic animal research predominantly used avian models. However, to account for the structural differences between avian and human eyes, the pigmented guinea pig has become a more preferred mammalian myopia model. It is generally accepted that abiochemical signal cascade originates in the retina and ends in the sclera. To identify proteins that initiate myopia development, this study investigated retinal protein expression using a novel proteomics approach. Most of the previous studies focused on the"single candidate" approach which limited the interaction among multiple candidates. The advancement of sequential window acquisition of all theoretical mass spectra (SWATH) in mass spectrometry (MS) has emerged as a more powerful liquid chromatography-based approach to study complex biological samples with the advantages of high throughput and reproducibility. Multiple-reaction monitoring (MRM)-based MS has been gradually adopted for biomarker verification in recent years. Considering antibodies of guinea pigs are not readily available and the high cost of conducting protein verification with antibody-dependent methods, this study optimized, applied SWATH-MS coupled with MRM-MS and a series of novel bioinformatics tools to screen and to confirm retinal proteins changes during myopia development. In the first stage, we successfully established a lens-induced myopia (LIM) model using-5D lens in guineapigs. Some instrumental performances were also optimized, such as liquid chromatography (LC) columns, LC gradients, and SWATH window setting. The cut-off threshold for protein quantitation was also determined. In the second stage, technological platforms of two-dimensional difference gel electrophoresis (2D DIGE) proteomics and SWATH-MS based proteomics were evaluated using the same retinal tissues. A mean of 732 protein spot pairs and 3,052 proteins were identified by the 2D DIGE and the SWATH-MS based proteomics, respectively. The 2D DIGE approach did not detect any differentially expressed proteins [>1.3-fold change (FC), P ≤0.05]. In contrast, the SWATH-MS based proteomics detected 58 differentially expressed proteins (FC of >1.20 or <0.83; P ≤0.05). SWATH-MS based proteomics outperformed the DIGE system in terms of sensitivity and throughput for detecting minor retinal protein changes. In the third stage, the whole retinal proteome and age-dependent changes in protein expression during emmetropization at postnatal days five (PN5), 17 (PN17), and 30 (PN30) were characterized by SWATH-MS based proteomics. A total of 4,020 proteins were identified as the largest guinea pig retina proteome, which was available on PeptideAtlas, PASS01289). Furthermore, 139 commonly regulated proteins (FC >1.20 or <0.83) and 31 commonly significant pathways were screened at the two more mature time points. The top 5 significant pathways were reported in previous myopic studies, including MAPK Signaling, EIF2 Signaling, Apoptosis Signaling, mTOR Signaling, and NGF Signaling.
In the fourth stage. SWATH-MS based proteomics was further performed in the guinea pigs after 2-day LIM,7-day LIM, and 2-day recovery. A total of 3,579 proteins were identified as a retinal ion library for SWATH quantitation (PASS01293). A total of 191, 136, and 110 differentially expressed proteins were found compared treated eyes to control eyes after 2-day LIM, 7-day LIM, and 2­day recovery respectively (FC>1.20 or<0.83, with a similar trend in two biological replicates). Translocase of outer mitochondrial membrane 6 (TOMM6)was the only protein commonly detected in all groups with expression >1.4 FC and showed bidirectional response between LIM and recovery. The upregulation of Argininosuccinate synthase 1 (ASS1) could promote the biosynthesis of arginine and then control myopia. In the fifth stage, the key biological pathways which potentially regulate retina signals in response to the optical defocus were screened using two commercially available bioinformatics software. Subsequently, we validated commonly regulated proteins and other index proteins that are strongly associated with key pathways using MRM-based targeted proteomics. The commonly expressed proteins (sentrin specific peptidase 7 and 5'-nucleotidase) across three time points were validated. By pathways analysis and confirming index proteins, we proposed the potential involvement of "TGF-β signaling", "PPAR signaling", and "HIPPO signaling pathway"in the early myopia/recovery paradigm. In conclusion, this study successfully characterized biometric and proteome changes during emmetropization in normal guinea pigs and myopic growth during LIM followed by recovery. We also demonstrated the feasibility of applying SWATH-MS based proteomics coupled with MRM-MS based protein target verification to characterize retinal proteins, quantify protein regulations, and investigate biochemical pathways at the systems biology level. Novel proteins and pathways involved in compensated myopic development were suggested by novel bioinformatics tools for the first time.
Subjects: Myopia
Hong Kong Polytechnic University -- Dissertations
Pages: xxxv, 412 pages : color illustrations
Appears in Collections:Thesis

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