A Comprehensive Profile of microRNAs in Human Tear Fluid
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE291021 | GEO | 2026/02/22
REPOSITORIES: GEO
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Project description:Keratin contamination is a common problem in mass spectrometry proteomic analyses, particularly in bottom-up mass spectrometry. The purpose of this study was to determine the protein contaminants introduced during the proteomic analysis of tear fluid.Human tear fluid samples were collected using Schirmer strips. Proteomic analyses were performed using liquid chromatography–tandem mass spectrometry (LC-MS/MS) on blank Schirmer strips and tear fluid samples, with empty vials serving as controls for assessing environmental contaminant proteins.This study revealed nine major contaminant keratins in the mass spectrometry analysis. Several other keratins were identified as constituents of tear fluid. Background subtraction is necessary for the accurate analysis of tear fluid using mass spectrometry.
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Project description:Male Non-Obese Diabetic (NOD) derived NOR mice develop autoimmune dacryoadenitis by 16 weeks of age including lymphocytic infiltration of the lacrimal glands, reduced tear production, and increased cysteine tear proteases. Unlike the NOD mice, the NOR mice do not develop diabetes and studies designed with the NOR mice are expected to not have diabetes development counfounding the results observed for Sjogren's development. Autoantigens with high levels of reactivity in serum and/or tears of diseased mice may have diagnostic potential. Serum was collected from mice at 16 weeks old male NOR mice along with age matched male healthy BALBc controls. Approximately 0.8 mL of blood was collected by cardiac puncture into serum separator tubes. Following cold centrifugation, serum was collected. Tears were collected following topical stimulation of the lacrimal gland with carbachol. For tear samples, tears were pooled from 1, 2 or 3 mice, respectively to generate three samples. This was done mainly to optimize the number of mice required to obtain sufficient sample for the assay as well as to assess the sensitivity of mouse tears for the proteinarray. 1 to 10 uL of tear or 10 uL of serum per sample was used to determine reactivity against 94 autoantigens using proteinarray.
2025-01-28 | GSE286502 | GEO
Project description:Fifty-six children and adolescents with type 1 diabetes at least one year after diagnosis, aged 6-17 years old and fifty-six healthy age- and sex-matched subjects were enrolled in this cross-sectional study. Tear samples were collected using Schirmer strips placed on the lower eyelid. The proteomic analysis was based on a detergent-assisted protein extraction and their digestion from the tears, analysis of the tryptic peptides with LC-MS/ enabling the identification, and quantification of the Shirmer strip protein content via DIA-NN, and subsequently the statistical and bioinformatic analysis using the R and Metascape enrichment analysis tool.
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Project description:Sjögren's Disease (SjD) is an autoimmune disease characterized by lymphocytic infiltration of salivary and lacrimal glands (LG). The LG produces the protein-rich aqueous component of tears, and SjD-associated autoimmune dacryoadenitis (AD) may thus alter tear autoantibody composition. The presence of tertiary lymphoid structures (TLS) in LG from two murine models of SjD-associated AD, male non-obese diabetic (NOD) and male non-obese insulitis resistant (NOR) mice, were evaluated using immunofluorescence. IgG and IgA reactivity in serum and tears from these models were probed in three studies against a panel of 80-120 autoantigens using autoantibody microarrays relative to serum and tears from healthy male BALB/c mice. Sources of Ig in tears were investigated using scRNA-Seq of the LG (GSE132420). Data were analyzed by R package Limma and Seurat. Analysis of immunofluorescence in LG sections from both SjD models showed TLS. Only one autoantibody was significantly elevated in tears and serum in both SjD models across all studies. Three autoantibodies were significantly elevated in serum but not in tears in both SjD models across all studies. Conversely, six IgG and thirteen IgA autoantibodies (6 sharing the same autoantigen) were significantly elevated in tears but not serum in both SjD models. Igha and Ighg2b expressing cells were identified in the plasma cell cluster of NOD.H2b LG. NOD and NOR mice with SjD-associated AD have distinct autoantibody profiles in tears and serum. Tear IgA isotype autoantibodies showed a greater diversity than tear IgG autoantibodies. TLS observed in LG are a likely source of the tear autoantibodies.
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Project description:Tears are a biological fluid that has diagnostic potential for ocular diseases. Extracellular vesicles (EVs), wildly detected in various biofluids including tears, are nanoparticles released by living cells and considered as promising detection sources for non-invasive liquid biopsy. Understanding the roles of tears and tear-EVs in ocular diseases such as dry eye can facilitate the studies of clinical diagnosis, which usually entails detecting such liquid objects with a rapid and effective method. In this study, we utilized a mass spectrometry based strategy to analyze peptidome/proteome profiles of tear and EVs for rapid dry eye diagnosis. Nano-sized EVs were isolated from tears of either healthy control (HC) individuals or dry eye syndrome (DES) patients, and the tear compositions were further analyzed by tracking their fingerprints with matrix-assisted laser desorption ionization/time-of-flight mass spectrometry (MALDI-TOF-MS). The fingerprints of tear-EVs could be observed in a dose-dependent manner as well as tears, allowing comparing their discriminant peaks between tears and EVs. By analyzing these peaks, the fingerprints of both tear and tear-EVs were showed to have a capability of distinguishing DES patients from HC donors, and providing an efficient way for screening potential DES biomarkers. The proposed tear and EV fingerprinting approach is expected to be a potential tool in rapid diagnosis of ocular disease and in-depth researches of pathogenesis.
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Project description:Purpose: Determination of inter- and intra-day variations in tear flow rate, tear fluid protein concentration as well as protein composition regarding their impact for future biomarker studies.Methods: Tear fluid was collected non-invasively from 18 healthy subjects performing Schirmer tests at four different time points repetitive in a period of two days. The tear flow rate with Schirmer test strips was measured. Proteins were extracted from strips and quantified using amino acid analysis. Protein composition was analyzed by data-independent (DIA) based mass spectrometry. To exclude any impairments to health, volunteers underwent a detailed neurological as well as an ophthalmological examination.Results: Whether tear fluid was collected from OS or OD did not affect the tear flow rate (p ≈ 0.63) or protein concentration (p ≈ 0.97) of individual subjects. Moreover, protein concentration was independent from the tear volume, so that a change in volume would only have an effect on total protein amount. When the examination days were compared, investigation of tear flow rate (p ≈ 0.001) and protein concentration (p ≈ 0.0003) indicated significant differences. Further mass spectrometric analysis of tear fluid revealed eleven differentially regulated proteins when comparing both examination days. Conclusion: Our findings provide evidence of inter-day variation in tear flow rate, tear proteome concentration and composition in healthy subjects, suggesting that inter-day variation need to be taken into consideration in biomarker research of tear fluid. Identified proteins were assigned to functions in the immune response, oxidative and reducing processes, as well as mannose metabolism.
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Project description:Protocol for tear fluid proteomics using Schirmer strips and LC-MS/MS.
2026-01-12 | PXD070688 | Pride
Project description:Tear fluid, a complex biofluid that contains thousands of proteins and can be collected non-invasively, has emerged as a promising source of biomarkers for ocular and systemic health. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is currently the primary method for discovering novel biomarkers in tear fluid. However, the method of tear collection can sig-nificantly impact LC-MS/MS analysis outcomes. Tear fluid is commonly collected using either Schirmer strips or capillary tubes. While capillary tubes offer distinct advantages, such as reduced extracellular contamination and reflex tearing, most LC-MS/MS protocol development has focused on optimizing protocols for Schirmer strips. This study addresses this gap by evaluating digestion protocols for tear fluid collected with capillary tubes, focusing on biomarker discovery using small-volume samples. In this study, we evaluated multiple digestion protocols for the shotgun quantitative LC-MS/MS analysis of small-volume tear fluid samples collected using glass capillary tubes. Protocol optimization was performed using pooled samples and then compared with the analysis of individual samples. Using the optimized protocol, 0.5μL were processed using a timsTOF Pro 2 mass spectrometer (Bruker) coupled online with an Evosep One liquid chroma-tography system (Evosep), leading to the identification of an average of 368 ± 87 proteins in pooled samples and 502 ± 127 proteins in individual small-volume tear fluid samples. This protocol highlights the practicality of using glass capillary tubes for comprehensive LC-MS/MS-based tear proteomics analysis, paving the way for detailed proteomics characterization of individual tear fluid samples rather than pooled samples. By shifting from pooled to individual samples, this approach greatly accelerates tear biomarker discovery, advancing precision and personalized medicine.
2025-05-07 | PXD060101 | Pride
Project description:Background: Mass spectrometry is a powerful technique for tear fluid proteomics, offering critical insights into its complex molecular composition. Data-dependent acquisition (DDA), the most commonly used approach, preferentially selects high-abundance proteins, limiting reproducibility and the quantification of low-abundance proteins. In contrast, data-independent acquisition (DIA) provides an unbiased and comprehensive proteomic profile by fragmenting all precursor ions, enhancing protein coverage, quantification accuracy and reproducibility. This study presents a comparative analysis of DDA and DIA approaches for tear fluid proteomics to improve detection of low-abundance proteins and facilitate biomarker discovery. Methods: Tear fluid samples were collected from healthy individuals using Schirmer strips, processed using in-strip protein digestion, and analyzed via liquid chromatography-tandem mass spectrometry (LC-MS/MS). DDA and DIA workflows were compared for proteomic depth, reproducibility, and data completeness. Quantification accuracy was assessed using serial dilutions of tear fluid samples in a complex biological matrix. Results: DIA identified 701 unique proteins and 2,444 peptides, significantly outperforming DDA, which identified 396 unique proteins and 1,447 peptides. DIA exhibited greater data completeness (78.7% proteins, 78.5% peptides) compared to DDA (42% proteins, 48% peptides) across replicates. Reproducibility was markedly improved in DIA, with median coefficients of variation (CVs) of 9.8% for proteins and 10.6% for peptides, compared to 17.3% and 22.3% in DDA, respectively. Quantification accuracy was also enhanced, demonstrating superior consistency across dilution series. Conclusion: This study demonstrates that DIA is a robust and reliable method for proteomic analysis of complex tear fluid samples, offering significantly greater depth, reproducibility, and accuracy compared to DDA.
2025-10-20 | PXD062423 | Pride
Project description:Background: Mass spectrometry is a powerful technique for tear fluid proteomics, offering critical insights into its complex molecular composition. Data-dependent acquisition (DDA), the most commonly used approach, preferentially selects high-abundance proteins, limiting reproducibility and the quantification of low-abundance proteins. In contrast, data-independent acquisition (DIA) provides an unbiased and comprehensive proteomic profile by fragmenting all precursor ions, enhancing protein coverage, quantification accuracy and reproducibility. This study presents a comparative analysis of DDA and DIA approaches for tear fluid proteomics to improve detection of low-abundance proteins and facilitate biomarker discovery. Methods: Tear fluid samples were collected from healthy individuals using Schirmer strips, processed using in-strip protein digestion, and analyzed via liquid chromatography-tandem mass spectrometry (LC-MS/MS). DDA and DIA workflows were compared for proteomic depth, reproducibility, and data completeness. Quantification accuracy was assessed using serial dilutions of tear fluid samples in a complex biological matrix. Results: DIA identified 701 unique proteins and 2,444 peptides, significantly outperforming DDA, which identified 396 unique proteins and 1,447 peptides. DIA exhibited greater data completeness (78.7% proteins, 78.5% peptides) compared to DDA (42% proteins, 48% peptides) across replicates. Reproducibility was markedly improved in DIA, with median coefficients of variation (CVs) of 9.8% for proteins and 10.6% for peptides, compared to 17.3% and 22.3% in DDA, respectively. Quantification accuracy was also enhanced, demonstrating superior consistency across dilution series. Conclusion: This study demonstrates that DIA is a robust and reliable method for proteomic analysis of complex tear fluid samples, offering significantly greater depth, reproducibility, and accuracy compared to DDA.
2025-10-20 | PXD062422 | Pride