Deep Spectral Library of Mice Retina for Myopia Research: Proteomics Dataset generated by SWATH and DIA-NN.

The retina plays a crucial role in processing and decoding visual information, both in normal development and during myopia progression. Recent advancements have introduced a library-independent approach for data-independent acquisition (DIA) analyses. This study demonstrates deep proteome identification and quantification in individual mice retinas during myopia development, with an average of 6,263 ± 86 unique protein groups. We anticipate that the use of a predicted retinal-specific spectral library combined with the robust quantification achieved within this dataset will contribute to a better understanding of the proteome complexity. Furthermore, a comprehensive mice retinal-specific spectral library was generated, encompassing a total identification of 9,401 protein groups, 70,041 peptides, 95,339 precursors, and 761,868 transitions acquired using SWATH-MS acquisition on a ZenoTOF 7600 mass spectrometer. This dataset surpasses the spectral library generated through high-pH reversed-phase fractionation by data-dependent acquisition (DDA). The data is available via ProteomeXchange with the identifier PXD046983. It will also serve as an indispensable reference for investigations in myopia research and other retinal or neurological diseases.

Metabolomics studies in common multifactorial eye disorders: a review of biomarker discovery for age-related macular degeneration, glaucoma, diabetic retinopathy and myopia.

Introduction: Multifactorial Eye disorders are a significant public health concern and have a huge impact on quality of life. The pathophysiological mechanisms underlying these eye disorders were not completely understood since functional and low-throughput biological tests were used. By identifying biomarkers linked to eye disorders, metabolomics enables early identification, tracking of the course of the disease, and personalized treatment. Methods: The electronic databases of PubMed, Scopus, PsycINFO, and Web of Science were searched for research related to Age-Related macular degeneration (AMD), glaucoma, myopia, and diabetic retinopathy (DR). The search was conducted in August 2023. The number of cases and controls, the study's design, the analytical methods used, and the results of the metabolomics analysis were all extracted. Using the QUADOMICS tool, the quality of the studies included was evaluated, and metabolic pathways were examined for distinct metabolic profiles. We used MetaboAnalyst 5.0 to undertake pathway analysis of differential metabolites. Results: Metabolomics studies included in this review consisted of 36 human studies (5 Age-related macular degeneration, 10 Glaucoma, 13 Diabetic retinopathy, and 8 Myopia). The most networked metabolites in AMD include glycine and adenosine monophosphate, while methionine, lysine, alanine, glyoxylic acid, and cysteine were identified in glaucoma. Furthermore, in myopia, glycerol, glutamic acid, pyruvic acid, glycine, cysteine, and oxoglutaric acid constituted significant metabolites, while glycerol, glutamic acid, lysine, citric acid, alanine, and serotonin are highly networked metabolites in cases of diabetic retinopathy. The common top metabolic pathways significantly enriched and associated with AMD, glaucoma, DR, and myopia were arginine and proline metabolism, methionine metabolism, glycine and serine metabolism, urea cycle metabolism, and purine metabolism. Conclusion: This review recapitulates potential metabolic biomarkers, networks and pathways in AMD, glaucoma, DR, and myopia, providing new clues to elucidate disease mechanisms and therapeutic targets. The emergence of advanced metabolomics techniques has significantly enhanced the capability of metabolic profiling and provides novel perspectives on the metabolism and underlying pathogenesis of these multifactorial eye conditions. The advancement of metabolomics is anticipated to foster a deeper comprehension of disease etiology, facilitate the identification of novel therapeutic targets, and usher in an era of personalized medicine in eye research.

A Protein Suspension-Trapping Sample Preparation for Tear Proteomics by Liquid Chromatography-Tandem Mass Spectrometry.

Tear fluid is one of the easily accessible biofluids that can be collected non-invasively. Tear proteomics has the potential to discover biomarkers for several ocular diseases and conditions. The suspension trapping column has been reported to be an efficient and user-friendly sample preparation workflow for the broad application of downstream proteomic analysis. Yet, this strategy has not been well-studied in the analysis of human tear proteome. The present protocol describes an integrated workflow from clinical human tear samples to purified peptides for non-invasive tear protein biomarker research using mass spectrometry, which provides insights into disease biomarkers and monitoring when combined with bioinformatics analysis. A protein suspension trapping sample preparation was applied and demonstrated the discovery of tear proteome with fast, reproducible, and user-friendly procedures, as a universal, optimized sample preparation for human tear fluid analysis. In particular, the suspension trapping procedure outperformed in-solution sample preparation in terms of peptide recovery, protein identification, and shorter sample preparation time.

Differential Responses of Retinal Neurons and Glia Revealed via Proteomic Analysis on Primary and Secondary Retinal Ganglion Cell Degeneration.

To explore the temporal profile of retinal proteomes specific to primary and secondary retinal ganglion cell (RGC) loss. Unilateral partial optic nerve transection (pONT) was performed on the temporal side of the rat optic nerve. Temporal and nasal retinal samples were collected at 1, 4 and 8 weeks after pONT (n = 4 each) for non-biased profiling with a high-resolution hybrid quadrupole time-of-flight mass spectrometry running on label-free SWATHTM acquisition (SCIEX). An information-dependent acquisition ion library was generated using ProteinPilot 5.0 and OneOmics cloud bioinformatics. Combined proteome analysis detected 2531 proteins with a false discovery rate of <1%. Compared to the nasal retina, 10, 25 and 61 significantly regulated proteins were found in the temporal retina at 1, 4, and 8 weeks, respectively (p < 0.05, FC ≥ 1.4 or ≤0.7). Eight proteins (ALDH1A1, TRY10, GFAP, HBB-B1, ALB, CDC42, SNCG, NEFL) were differentially expressed for at least two time points. The expressions of ALDH1A1 and SNCG at nerve fibers were decreased along with axonal loss. Increased ALDH1A1 localization in the inner nuclear layer suggested stress response. Increased GFAP expression demonstrated regional reactivity of astrocytes and Muller cells. Meta-analysis of gene ontology showed a pronounced difference in endopeptidase and peptidase inhibitor activity. Temporal proteomic profiling demonstrates established and novel protein targets associated with RGC damage.

Additive effects of narrowband light and optical defocus on chick eye growth and refraction.

BACKGROUND: In the past decade and during the COVID pandemic, the prevalence of myopia has reached epidemic proportions. To address this issue and reduce the prevalence of myopia and its complications, it is necessary to develop more effective interventions for controlling myopia. In this study, we investigated the combined effects of narrowband lights and competing defocus on eye growth and refraction in chicks, an important step in understanding the potential for these interventions to control myopia. This is the first time these effects have been characterized. METHODS: Three groups of five-day-old chicks (n = 8 per group) were raised in three different lighting conditions: white, red, and blue for 13 days in a 12/12-h light/dark diurnal cycle. One eye was randomly selected for applications of a dual-power optical lens (- 10 D/ + 10 D, 50∶50), while another eye was left untreated as control. Vitreous chamber depth (VCD), axial length (AL), choroidal thickness (CT) and refractive errors were measured at pre-exposure (D0) and following 3 (D3), 7 (D7), 10 (D10), and 13 days (D13) of light exposure. RESULTS: Under white light, the dual-power lens induced a hyperopic shift [at D13, mean spherical equivalent refraction (SER), treated vs. control: 4.81 ± 0.43 D vs. 1.77 ± 0.21 D, P < 0.001] and significantly reduced the progression of axial elongation (at D13, change in AL, treated vs. control: 1.25 ± 0.04 mm vs. 1.45 ± 0.05 mm, P < 0.01). Compared to white light alone, blue light alone induced a hyperopic shift (at D13, mean SER, blue vs. white: 2.75 ± 0.21 D vs. 1.77 ± 0.21 D, P < 0.01) and significantly reduced axial elongation (at D13, change in AL, blue vs. white: 1.17 ± 0.06 mm vs. 1.45 ± 0.05 mm, P < 0.01) in control eyes. When comparing all conditions, eyes exposed to blue light plus dual-power lens had the least axial elongation (at D13, change in AL, 0.99 ± 0.05 mm) and were the most hyperopic (at D13, mean SER, 6.36 ± 0.39 D). CONCLUSIONS: Both narrowband blue light and dual-power lens interventions were effective in inducing a hyperopic shift in chicks, and provided protection against myopia development. The combination of these interventions had additive effects, making them potentially even more effective. These findings support the use of optical defocus interventions in combination with wavelength filters in clinical studies testing their effectiveness in treating myopia in children.

Mechanistic Effects of Baicalein on Aqueous Humor Drainage and Intraocular Pressure.

Elevated intraocular pressure (IOP) is a major risk factor for glaucoma that results from impeded fluid drainage. The increase in outflow resistance is caused by trabecular meshwork (TM) cell dysfunction and excessive extracellular matrix (ECM) deposition. Baicalein (Ba) is a natural flavonoid and has been shown to regulate cell contraction, fluid secretion, and ECM remodeling in various cell types, suggesting the potential significance of regulating outflow resistance and IOP. We demonstrated that Ba significantly lowered the IOP by about 5 mmHg in living mice. Consistent with that, Ba increased the outflow facility by up to 90% in enucleated mouse eyes. The effects of Ba on cell volume regulation and contractility were examined in primary human TM (hTM) cells. We found that Ba (1-100 µM) had no effect on cell volume under iso-osmotic conditions but inhibited the regulatory volume decrease (RVD) by up to 70% under hypotonic challenge. In addition, Ba relaxed hTM cells via reduced myosin light chain (MLC) phosphorylation. Using iTRAQ-based quantitative proteomics, 47 proteins were significantly regulated in hTM cells after a 3-h Ba treatment. Ba significantly increased the expression of cathepsin B by 1.51-fold and downregulated the expression of D-dopachrome decarboxylase and pre-B-cell leukemia transcription factor-interacting protein 1 with a fold-change of 0.58 and 0.40, respectively. We suggest that a Ba-mediated increase in outflow facility is triggered by cell relaxation via MLC phosphorylation along with inhibiting RVD in hTM cells. The Ba-mediated changes in protein expression support the notion of altered ECM homeostasis, potentially contributing to a reduction of outflow resistance and thereby IOP.

Alteration of EIF2 Signaling, Glycolysis, and Dopamine Secretion in Form-Deprived Myopia in Response to 1% Atropine Treatment: Evidence From Interactive iTRAQ-MS and SWATH-MS Proteomics Using a Guinea Pig Model.

Purpose: Atropine, a non-selective muscarinic antagonist, effectively slows down myopia progression in human adolescents and several animal models. However, the underlying molecular mechanism is unclear. The current study investigated retinal protein changes of form-deprived myopic (FDM) guinea pigs in response to topical administration of 1% atropine gel (10 g/L). Methods: At the first stage, the differentially expressed proteins were screened using fractionated isobaric tags for a relative and absolute quantification (iTRAQ) approach, coupled with nano-liquid chromatography-tandem mass spectrometry (nano-LC-MS/MS) (n = 24, 48 eyes) using a sample pooling technique. At the second stage, retinal tissues from another cohort with the same treatment (n = 12, 24 eyes) with significant ocular changes were subjected to label-free sequential window acquisition of all theoretical mass spectra (SWATH-MS) proteomics for orthogonal protein target confirmation. The localization of Alpha-synuclein was verified using immunohistochemistry and confocal imaging. Results: A total of 1,695 proteins (8,875 peptides) were identified with 479 regulated proteins (FC ≥ 1.5 or ≤0.67) found from FDM eyes and atropine-treated eyes receiving 4-weeks drug treatment using iTRAQ-MS proteomics. Combining the iTRAQ-MS and SWATH-MS datasets, a total of 29 confident proteins at 1% FDR were consistently quantified and matched, comprising 12 up-regulated and 17 down-regulated proteins which differed between FDM eyes and atropine treated eyes (iTRAQ: FC ≥ 1.5 or ≤0.67, SWATH: FC ≥ 1.4 or ≤0.71, p-value of ≤0.05). Bioinformatics analysis using IPA and STRING databases of these commonly regulated proteins revealed the involvement of the three commonly significant pathways: EIF2 signaling; glycolysis; and dopamine secretion. Additionally, the most significantly regulated proteins were closely connected to Alpha-synuclein (SNCA). Using immunostaining (n = 3), SNCA was further confirmed in the inner margin of the inner nuclear layer (INL) and spread throughout the inner plexiform layer (IPL) of the retina of guinea pigs. Conclusion: The molecular evidence using next-generation proteomics (NGP) revealed that retinal EIF2 signaling, glycolysis, and dopamine secretion through SNCA are implicated in atropine treatment of myopia in the FDM-induced guinea pig model.

Human tear proteome dataset in response to daily wear of water gradient contact lens using SWATH-MS approach.

Water Gradient Contact Lens (WGCL) is a new generation material that combines the benefits of Silicone hydrogel (SiHy) and traditional hydrogel contact lenses by modifying the materials between the core and the surface. However, its impact on tear proteome has not been explored. Tears were collected on healthy young adults using Schirmer's strip at baseline, 1-week, and 1-month of WGCL lens wear (n=15) and age-matched untouched controls (n=10). Equal amounts of tears samples from individuals of WGCL and control groups were randomly pooled to form representative equal parts at each condition (n=3 for WGCL wear and age-matched untouched control group) at each condition (baseline, 1-week, and 1-month). Tears were prepared using the S-Trap sample preparation followed by the analysis of a TripleTOF 6600 mass spectrometer. Using Information-dependent acquisition (IDA), a total of 725 tear proteins (6760 distinct peptides) were identified in the constructed spectral library at 1% FDR. Using data-independent acquisition (SWATH-MS), data were analyzed and processed using PeakView (v2.2, SCIEX), with the top differentially expressed proteins at each time point (baseline, 1-week, and 1-month) presented. All acquired raw data (IDA and SWATH-MS) were submitted and published on the Peptide Atlas public repository (http://www.peptideatlas.org/) for general release (Data ID PASS01589).

Functional connexin35 increased in the myopic chicken retina.

Our previous research showed that increased phosphorylation of connexin (Cx)36 indicated extended  coupling of AII amacrine cells (ACs) in the rod-dominant mouse myopic retina. This research will determine whether phosphorylation at serine 276 of Cx35-containing gap junctions increased in the myopic chicken, whose retina is cone-dominant. Refractive errors and ocular biometric dimensions of 7-days-old chickens were determined following 12 h and 7 days induction of myopia by a -10D lens. The expression pattern and size of Cx35-positive plaques were examined in the early (12 h) and compensated stages (7 days) of lens-induced myopia (LIM). At the same time, phosphorylation at serine 276 (functional assay) of Cx35 in strata 5 (S5) of the inner plexiform layer was investigated. The axial length of the 7 days LIM eyes was significantly longer than that of non-LIM controls (P < 0.05). Anti-phospho-Ser276 (Ser276-P)-labeled plaques were significantly increased in LIM retinas at both 12 h and 7 days. The density of Ser276-P of Cx35 was observed to increase after 12 h LIM. In the meanwhile, the areas of existing Cx35 plaques did not change. As there was more phosphorylation of connexin35 at Ser276 at both the early and late stages (12 h) and 7 days of LIM chicken retinal activity, the coupling with ACs could be increased in myopia development of the cone-dominated chicken retina.

SWATH Based Quantitative Proteomics Reveals Significant Lipid Metabolism in Early Myopic Guinea Pig Retina.

Most of the previous myopic animal studies employed a single-candidate approach and lower resolution proteomics approaches that were difficult to detect minor changes, and generated limited systems-wide biological information. Hence, a complete picture of molecular events in the retina involving myopic development is lacking. Here, to investigate comprehensive retinal protein alternations and underlying molecular events in the early myopic stage, we performed a data-independent Sequential Window Acquisition of all Theoretical Mass Spectra (SWATH) based proteomic analysis coupled with different bioinformatics tools in pigmented guinea pigs after 4-day lens-induced myopia (LIM). Myopic eyes compared to untreated contralateral control eyes caused significant changes in refractive error and choroid thickness (p < 0.05, n = 5). Relative elongation of axial length and the vitreous chamber depth were also observed. Using pooled samples from all individuals (n = 10) to build a species-specific retinal ion library for SWATH analysis, 3202 non-redundant proteins (with 24,616 peptides) were identified at 1% global FDR. For quantitative analysis, the 10 individual retinal samples (5 pairs) were analyzed using a high resolution Triple-TOF 6600 mass spectrometry (MS) with technical replicates. In total, 37 up-regulated and 21 down-regulated proteins were found significantly changed after LIM treatment (log2 ratio (T/C) > 0.26 or < -0.26; p ≤ 0.05). Data are accepted via ProteomeXchange with identifier PXD025003. Through Ingenuity Pathways Analysis (IPA), "lipid metabolism" was found as the top function associated with the differentially expressed proteins. Based on the protein abundance and peptide sequences, expression patterns of two regulated proteins (SLC6A6 and PTGES2) identified in this pathway were further successfully validated with high confidence (p < 0.05) using a novel Multiple Reaction Monitoring (MRM) assay on a QTRAP 6500+ MS. In summary, through an integrated discovery and targeted proteomic approach, this study serves as the first report to detect and confirm novel retinal protein changes and significant biological functions in the early LIM mammalian guinea pigs. The study provides new workflow and insights for further research to myopia control.

Corneal proteome and differentially expressed corneal proteins in highly myopic chicks using a label-free SWATH-MS quantification approach.

Myopia, or short-sightedness, is a highly prevalent refractive disorder in which the eye's focal length is too short for its axial dimension in its relaxed state. High myopia is associated with increased risks of blinding ocular complications and abnormal eye shape. In addition to consistent findings on posterior segment anomalies in high myopia (e.g., scleral remodeling), more recent biometric and biomechanical data in myopic humans and animal models also indicate anterior segment anomalies (e.g., corneal biomechanical properties). Because the cornea is the anterior-most ocular tissue, providing essential refractive power and physiological stability, it is important to understand the biochemical signaling pathway during myopia development. This study first aimed to establish the entire chicken corneal proteome. Then, using the classical form deprivation paradigm to induce high myopia in chicks, state-of-the-art bioinformatics technologies were applied to identify eight differentially expressed proteins in the highly myopic cornea. These results provide strong foundation for future corneal research, especially those using chicken as an animal model for myopia development.

High-pH reversed-phase fractionated neural retina proteome of normal growing C57BL/6 mouse.

The retina is a key sensory tissue composed of multiple layers of cell populations that work coherently to process and decode visual information. Mass spectrometry-based proteomics approach has allowed high-throughput, untargeted protein identification, demonstrating the presence of these proteins in the retina and their involvement in biological signalling cascades. The comprehensive wild-type mouse retina proteome was prepared using a novel sample preparation approach, the suspension trapping (S-Trap) filter, and further fractionated with high-pH reversed phase chromatography involving a total of 28 injections. This data-dependent acquisition (DDA) approach using a Sciex TripleTOF 6600 mass spectrometer identified a total of 7,122 unique proteins (1% FDR), and generated a spectral library of 5,950 proteins in the normal C57BL/6 mouse retina. Data-independent acquisition (DIA) approach relies on a large and high-quality spectral library to analyse chromatograms, this spectral library would enable access to SWATH-MS acquisition to provide unbiased, multiplexed, and quantification of proteins in the mouse retina, acting as the most extensive reference library to investigate retinal diseases using the C57BL/6 mouse model.

Combined retinal proteome datasets in response to atropine treatment using iTRAQ and SWATH-MS based proteomics approaches in guinea pig myopia model.

Atropine, a non-selective muscarinic antagonist, is known to slow down myopia progression in human adolescents and in several animal models. However, its underlying molecular mechanism is unclear. The present work built a monocular form-deprivation myopia (FDM) guinea pig model, using facemasks as well as atropine treatment on FDM eyes for 2 and 4 weeks. Retinal protein changes in response to the FDM and effects of topical administration of atropine were screened for the two periods using fractionated isobaric tags for a relative and absolute quantification (iTRAQ) approach coupled with nano-liquid chromatography-tandem mass spectrometry (nano-LC-MS/MS) (n=24, 48 eyes). Retinal tissues from another cohort receiving 4-weeks FDM with atropine treatment (n=12, 24 eyes) with more significant changes were subjected to sequential window acquisition of all theoretical mass spectra (SWATH-MS) proteomics for further protein target confirmation. A total of 1695 proteins (8875 peptides) and 5961 proteins (51871 peptides) were identified using iTRAQ and SWATH approaches, respectively. Using the Paragon algorithm in the ProteinPilotTM software, the three most significantly up-regulated and down-regulated proteins that were commonly found in both ITRAQ and SWATH experiments are presented. All raw data generated from the work were submitted and published in the Peptide Atlas public repository (http://www.peptideatlas.org/) for general release (Data ID PASS01507).

Data on protein changes of chick vitreous during normal eye growth using data-independent acquisition (SWATH-MS).

Myopia is the most common refractive error which is estimated to affect half the population of the world by 2050. It has been suggested that it could be determined by multiple factors such as environmental and genetic, but the mechanism behind the cause of myopia is still yet to be identified. Vitreous humor (VH) is a transparent gelatin-like substance that takes up to 80% of the volume of the eye, making it the largest component of the eye. Although VH is the main contributor to axial elongation of the eye including normal eye growth (emmetropization) and myopia, the diluted nature of VH (made up of 99% of water) made it difficult for less abundant molecules to be identified and therefore often overlooked. Using the more sensitive label-free mass spectrometry approach with data-independent acquisition (SWATH-MS), we established a comprehensive VH proteome library in chick animal model and quantified possible protein biomarkers that are responsible for the axial elongation during emmetropization (7, 14, 21, 28 days after hatching, n = 48 eyes). Raw data files for both information-dependent acquisition (IDA) and data-independent acquisition (SWATH-MS) were uploaded on PeptideAtlas for public access (http://www.peptideatlas.org/PASS/PASS01258).

Alteration of retinal metabolism and oxidative stress may implicate myopic eye growth: Evidence from discovery and targeted proteomics in an animal model.

Myopia, the most common cause of impaired vision, may induce sight- threatening diseases or ocular complications due to axial elongation. The exact mechanisms underlying myopia development have received much attention and understanding of these is necessary for clinical prevention or therapeutics. In this study, quantitative proteomics using Isotope Coded Protein Label (ICPL) was applied to identify differentially regulated proteins in the retinas of myopic chicks and, from their presence, infer the possible pathogenesis of excessive ocular elongation. Newly hatched white leghorn chicks (n = 15) wore -10D and + 10D lenses bilaterally for 3 and 7 days, respectively, to develop progressive lens-induced myopia (LIM) and hyperopia (LIH). Retinal proteins were quantified with nano-liquid chromatography electrospray ionization coupled with tandem mass spectrometry (nanoLC-ESI-MS/MS). Bioinformatics analysis of differentially regulated proteins revealed that the majority originated from the cytoplasmic region and were related to various metabolic, glycolytic, or oxidative processes. The fold changes of four proteins of interest (vimentin, apolipoprotein A1, interphotoreceptor retinoid binding protein, and glutathione S-transferase) were further confirmed by a novel high-resolution multiple reaction monitoring mass spectrometry (MRM-HR) using a label-free approach. SIGNIFICANCE: Discovery of effective protein biomarkers of myopia has been extensively studied to inhibit myopia progression. This study first applied lens-induced hyperopia and myopia in the same chick to maximize the inter-ocular differences, aiming to discover more protein biomarker candidates. The findings provided new evidence that myopia was metabolism related, accompanied by altered energy generation and oxidative stress at retinal protein levels. The results in the retina were also compared to our previous study in vitreous using ICPL quantitative technology. We have now presented the protein changes in these two adjacent tissues, which may provide extra information of protein changes during ocular growth in myopia.

Data on corneal proteome and differentially expressed corneal proteins in highly myopic chicks using a data independent quantification approach.

Myopia is an abnormal refractive status, explained by an excessive ocular lengthening mostly in posterior segments. Although growing evidence of anterior segments, specifically altered corneal geometries with biomechanical properties in myopes have been reported, the mechanism behind is poorly understood. We hereby prepared experimentally induced highly myopic chicks to investigate the molecular basis of corneal remodeling by applying a novel proteomic approach integrated with information dependent acquisition (IDA) and data independent quantification (SWATH-MS) analysis. As a result, differentially expressed protein biomarkers that might be involved in structural changes were screened based on the first of its kind unique chicken corneal proteome. All generated raw data from IDA and SWATH-MS are accessible at Peptide Atlas public repository (http://www.peptideatlas.org/PASS/PASS01410) for general release.

Quantitative profiling of regional protein expression in rat retina after partial optic nerve transection using fluorescence difference two­dimensional gel electrophoresis.

To examine the difference between primary and secondary retinal ganglion cell (RGC) degeneration, the protein expression at four regions of retina including superior, temporal, inferior and nasal quadrant in a rat model of partial optic nerve transection (pONT) using 2­D Fluorescence Difference Gel Electrophoresis (DIGE) were investigated. Unilateral pONT was performed on the temporal side of optic nerves of adult Wistar rats to separate primary and secondary RGC loss. Topographical quantification of RGCs labeled by Rbpms antibody and analysis of axonal injury by grading of optic nerve damage at 1 week (n=8) and 8 weeks (n=15) after pONT demonstrated early RGC loss at temporal region, which is considered as primary RGC degeneration and progressing RGC loss at nasal region, which is considered as secondary RGC degeneration. Early protein expression in each retinal quadrant (n=4) at 2 weeks after pONT was compared with the corresponding quadrant in the contralateral control eye by DIGE. For all comparisons, 24 differentially expressed proteins (>1.2­fold; P<0.05; ≥3 non­duplicated peptide matches) were identified by mass spectrometry (MS). Interestingly, in the nasal retina, serum albumin and members of crystallin family, including αA, αB, ßA2, ßA3, ßB2 and gamma S indicating stress response were upregulated. By contrast, only αB and ßA2 crystallin proteins were altered in temporal quadrant. In the superior and inferior quadrants, ßB2 crystallin, keratin type I, S­arrestin and lamin­B1 were upregulated, while heat shock cognate 71 kDa protein and heterogeneous nuclear ribonucleoproteins A2/B1 were downregulated. In summary, the use of DIGE followed by MS is useful to detect early regional protein regulation in the retina after localized optic nerve injury.

Data on differentially expressed proteins in retinal emmetropization process in guinea pig using integrated SWATH-based and targeted-based proteomics.

Myopia is generally regarded as a failure of normal emmetropization process, however, its underlying molecular mechanisms are unclear. Retinal protein profile changes using integrated SWATH and MRM-HR MS were studied in guinea pigs at 3- and 21-days of age, where the axial elongation was significantly detected. Differential proteins expressions were identified, and related to pathways which are important in postnatal development in retina, proliferation, breakdown of glycogen-energy and visual phototransduction. These results are significant as key retinal protein players and pathways that underlying emmetropization can be discovered. All raw data generated from IDA and SWATH acquisitions were accepted and published in the Peptide Atlas public repository (http://www.peptideatlas.org/) for general release (Data ID PASS00746). A more comprehensive analysis of this data can be obtained in the article "Integrated SWATH-based and targeted-based proteomics provide insights into the retinal emmetropization process in guinea pig" in Journal of Proteomics (Shan et al., 2018) [1].

Proteomic analysis of chick retina during early recovery from lens­induced myopia.

Myopia development has been extensively studied from different perspectives. Myopia recovery is also considered important for understanding the development of myopia. However, despite several previous studies, retinal proteomics during recovery from myopia is still relatively unknown. Therefore, the aim of the present study was to investigate the changes in protein profiles of chicken retinas during early recovery from lens­induced myopia to evaluate the signals involved in the adjustment of this refractive disorder. Three­day old chickens wore glasses for 7 days (­10D lens over the right eye and a plano lens as control over the left eye), followed by 24 h without lenses. Protein expression in the retina was measured by two­dimensional fluorescence difference gel electrophoresis (2D­DIGE). Pro­Q Diamond phosphoprotein staining 2D gel electrophoresis was used to analyze phosphoprotein profiles. Protein spots with significant differences (P<0.05) were analyzed by mass spectrometry. The minus lens­treated eye became myopic, however following 24 h recovery, less myopia was observed. 2D­DIGE proteomic analysis demonstrated that three identified protein spots were upregulated at least 1.2­fold in myopic recovery retinas compared with those of the controls, Ras related protein Rab­11B, S­antigen retina and pineal gland and 26S proteasome non­ATPase regulatory subunit 14. Pro­Q Diamond images further revealed three protein spots with significant changes (at least 1.8­fold): ß­tubulin was downregulated, while peroxiredoxin 4 and ubiquitin carboxyl­terminal hydrolase­L1 were upregulated in the recovery retinas compared with the control eye retinas. The present study detected previously unreported protein changes in recovering eyes, therefore revealing their potential involvement in retinal remodeling during eye ball reforge.