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Mass spectrometric detection of keratins in tear fluid

ABSTRACT: 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.

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Saleh Ahmed

LAB HEAD: Ashok Sharma

PROVIDER: PXD061409 | Pride | 2025-03-03

REPOSITORIES: Pride

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			Action	DRS
	Blank-strip-1.msf	Msf
	Blank-strip-1.raw	Raw
	Blank-strip-2.msf	Msf
	Blank-strip-2.raw	Raw
	Blank-strip-3.msf	Msf

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Publications

Mass spectrometric detection of keratins in tear fluid.

Ahmed Saleh S Altman Jeremy J Jones Garrett G Lee Tae Jin TJ Robertson Danielle M DM Zhi Wenbo W Sharma Shruti S Sharma Ashok A

Experimental eye research 20250104

<h4>Purpose</h4>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.<h4>Methods</h4>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 em ...[more]

PMID: 39761842

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Investigation of inter- and intra-day variability of tear fluid regarding flow rate, protein concentration as well as protein composition

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.

2023-10-24 | PXD037811 | Pride

Systematic Investigation of Targeted Analysis of Metabolites and Proteins in Tear Fluid

Project description:Since the sampling of tear fluid seems rather variable we sought to evaluate a protocol for combined metabolomic and proteomic analyses with regard to clinical applicability. Tear fluid was taken from twelve human volunteers at different time points using specially designed Schirmer strips. Following liquid extraction of metabolites from standardized punchesthe remaining material was processed for bottom-up proteomics. The targeted proteomic analysis of 15 tear proteins revealed a mean intra-individual variation of 23 % merely for the three most abundant proteins. Overall, our newly established workflow opens perspectives for clinical and routine diagnostic applications using well accessible tear fluid.

2019-11-12 | PXD008536 | Pride

Proteomic Analysis of Human Conjunctival Cells and Tears Collected with Schirmer Strips Using TimsTOF Pro

Project description:This study aimed to investigate the human tear proteins extracted from whole (W) Schirmer strips (ScS) and their two parts, the bulb (B) and the rest of the strip (R) with a comprehensive proteomic approach using a trapped ion mobility mass spectrometer, the timsTOF Pro. Eight ScS were collected from two healthy subjects at 4 different visits to be separated into three batches: 4W, 4B and 4R. In total, 1582 proteins were identified in the W, B and R batches. Among all iden-tified proteins, binding proteins (43.4%) and those with catalytic activity (42.2%) constituted more than 80% of the molecular functions. The most represented biological processes were cel-lular processes (31.2%), metabolic processes (20.8%) and biological regulation (13.1%). Enzymes were the most represented protein class (41 %), consisting mainly of hydrolases (47.5%), oxi-doreductases (22.1%) and transferases (16.7%). The bulb (B), which is in contact with the conjunc-tiva, might collect both tear and cell proteins and therefore promote identification of more pro-teins. Processing B and R separately before MS analysis, combined with the high data acquisition speed and the addition of ion-mobility-based separation in timsTOF Pro, can bring a new dimension to biomarker investigations of a limited sample such as tear fluid.

2022-01-27 | PXD030334 | Pride

Comparison of Different Mass-Spectrometry Workflows for the Proteomic Analysis of Tear Fluid

Project description:Proteomic analysis of tear fluid is challenging mainly due to a wide dynamic range of proteins and small sample volume. However, recent advancements in mass spectrometry have revolutionized the field of proteomics through its unprecedented depth, speed, and accuracy even with small sample volumes. In this study using the Orbitrap Fusion Tribrid mass spectrometer, we compared four different mass-spectrometry workflows for proteomic analysis of human tear fluid.

2022-04-04 | PXD030990 | Pride

Tear Proteomics Analysis in Children and Adolescents with Type 1 Diabetes

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.

2024-10-17 | PXD052994 | Pride

microRNA expression data from human corneal epithelial cells exposed to media, human tear, media + P. aeruginosa antigens, tear + P. aeruginosa antigens

Project description:Exposure to tear fluid can enhance corneal epithelial cells' ability to resist bacterial virulence mechanisms by upregulating epithelial-derived innate defense genes. The aim of this study was to further elucidate the mechanisms by which tear fluid modulates epithelial cell susceptibility to P. aeruginosa internalization and the relationship to known to be upregulated genes with tear fluid exposure. The hypothesis tested was that tear fluid effects on epithelial cells involve the induction of microRNA expression to modify innate defense gene responses to bacterial challenge. Human corneal epithelial cells were incubated in either 40 ul of fresh human tear fluid or high calcium KGM without antibiotics for 16 hours before extraction or before incubation with P. aeruginosa antigens for 3 h then extraction.

2012-07-13 | E-GEOD-39341 | biostudies-arrayexpress

Human Tear using MALDI-TOF-MS for Dry Eye syndrome

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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microRNA expression data from human corneal epithelial cells exposed to media, human tear, media + P. aeruginosa antigens, tear + P. aeruginosa antigens

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