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.

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).

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.

The Effect of Low-Dose Atropine on Alpha Ganglion Cell Signaling in the Mouse Retina.

Low-dose atropine helps to control myopia progression with few side effects. However, the impact of atropine, a non-selective muscarinic Acetylcholine (ACh) receptor antagonist, on retinal ganglion cells (RGCs) remains unclear. After immersing the cornea and adjacent conjunctiva of enucleated eyes in 0.05% (approximately 800 µM) atropine solution for 30 min, the atropine concentration reached in the retina was below 2 µM. After direct superfusion of the retina with 1 µM atropine (considering that the clinical application of 0.05% atropine eye drops will be diluted over time due to tear flow for 30 min), no noticeable changes in the morphology of ON and OFF alpha RGCs (αRGCs) were observed. Atropine affected the light-evoked responses of ON and OFF αRGCs in a dose- and time-dependent fashion. Direct application of less than 100 µM atropine on the retina did not affect light-evoked responses. The time latency of light-induced responses of ON or OFF αRGCs did not change after the application of 0.05-100 µM atropine for 5 min. However, 50 µM atropine extended the threshold of joint inter-spike interval (ISI) distribution of the RGCs. These results indicated that low-dose atropine (<0.5 µM; equal to 1% atropine topical application) did not interfere with spike frequency, the pattern of synchronized firing between OFF αRGCs, or the threshold of joint ISI distribution of αRGCs. The application of atropine unmasked inhibition to induce ON responses from certain OFF RGCs, possibly via the GABAergic pathway, potentially affecting visual information processing.

Data on assessment of safety and tear proteome change in response to orthokeratology lens - Insight from integrating clinical data and next generation proteomics.

Breath-O™ Correct Ortho-K lenses are newly designed ortho-K lenses which are made from a silicon and fluoride containing methacrylate compound. This compound is said to be more flexible, durable and less likely to break compared to traditional Ortho-K lenses. The special design of this Ortho-K lens can reshape the corneal profile to induce temporary myopic reduction while producing beneficial peripheral hyperopic defocus for myopia control. To evaluate the safety and ocular surface responses of overnight Ortho-K wear over 1 and 3 months using this new type of material, we evaluated the clinical parameters (corneal integrity, corneal biomechanics, corneal endothelial health, non-invasive keratographical break-up time) and profiled the change of global tear proteome on healthy young subjects using next generation proteomics (SWATH-MS). The acquired mass spectrometric data were processed and analyzed using a cloud based Oneomics™ bioinformatic platform. All raw data generated from Information-dependent acquisition (IDA) and SWATH acquisitions were accepted and published in the Peptide Atlas public repository for general release (http://www.peptideatlas.org/PASS/PASS01367).