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

ABSTRACT: 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.

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Tear

SUBMITTER: Ashok Sharma

LAB HEAD: Ashok Sharma

PROVIDER: PXD030990 | Pride | 2022-04-04

REPOSITORIES: Pride

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Dataset's files

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			Action	DRS
	T017_Post-extraction_digestion_CID.msf	Msf
	T017_Post-extraction_digestion_CID.raw	Raw
	T017_Post-extraction_digestion_HCD.msf	Msf
	T017_Post-extraction_digestion_HCD.raw	Raw
	T017_in-strip_digestion_CID.msf	Msf

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Publications

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

Jones Garrett G Lee Tae Jin TJ Glass Joshua J Rountree Grace G Ulrich Lane L Estes Amy A Sezer Mary M Zhi Wenbo W Sharma Shruti S Sharma Ashok A

International journal of molecular sciences 20220219 4

The tear film is a multi-layer fluid that covers the corneal and conjunctival epithelia of the eye and provides lubrication, nutrients, and protection from the outside environment. Tear fluid contains a high concentration of proteins and has thus been recognized as a potential source of biomarkers for ocular disorders due to its proximity to disease sites on the ocular surface and the non-invasive nature of its collection. This is particularly true in the case of dry eye disease, which directly ...[more]

PMID: 35216421

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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

Putative tear fluid biomarkers of diabetic retinopathy

Project description:Identification of tear fluid biomarkers may offer a non-invasive strategy for detecting diabetic patients with increased risk of developing diabetic retinopathy (DR) or increased disease progression, thus helping both improving diagnostic accuracy and understanding the pathophysiology of the disease. The goal of this study was to characterize the proteomic profile of human tear fluid and examine changes in proteins expression in different stages of diabetic retinopathy.

2022-05-31 | PXD033101 | Pride

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

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.

2020-08-20 | PXD020217 | 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

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

2012-07-14 | GSE39341 | GEO

Human tear fluid proteome dataset for usage as a spectral library and for protein modeling

Project description:This article provides a detailed dataset of human tear fluid proteins. Samples were fractionated by sodium dodecyl sulfate (SDS) gel electrophoresis resulting in 48 fractions that were spiked with an indexed retention time (iRT) peptide standard. These data are based on a data-dependent acquisition (DDA) mass spectrometric approach and can be used for example as a spectral library for tear fluid proteome analysis by data-independent acquisition (DIA). Moreover, the provided data set can be used with optimized HPLC as well as mass spectrometric settings for proteins/peptides of interest. Besides these aspects, this dataset can serve as a protein overview for gene ontology enrichment analysis as well as for modeling and benchmarking of multiple signaling pathways associated with the ocular surface in healthy or disease stages.

2019-08-15 | PXD011075 | Pride

Tear proteomics of young patients wearing glasses, orthokeratology lenses, and soft contact lenses

Project description:Contact lens-related ocular surface complications occur more often in teenagers and young adults. The purpose of this study was to determine changes in tear proteome of young patients wearing glasses (GL), orthokeratology lenses (OK), and soft contact lenses (SCL). Twenty-two young patients (10-26 years of age) who were established (> 3 years) GL (n=10), OK (n=6), and SCL (n=6) wearers were recruited. Tears were collected via Schirmer strips. Proteomic data were collected using a data-independent acquisition-parallel accumulation serial fragmentation (diaPASEF) workflow. Tear proteome composition and abundance were compared across different correction groups and between the children (age <18 years) and young adults (age 18 years) GL wearers. We identified 3406 protein groups in tears. Among proteins identified in 80% of tear samples, 8 proteins were upregulated, and 11 proteins were down-regulated in the SCL group compared to the OK group. Eighty-two proteins were differentially expressed in children and young adults GL wearers, among which 67 proteins were upregulated, and 15 proteins were downregulated in children. These 82 proteins were involved in 1 inflammation, 9 immune, and 1 glycoprotein metabolic biological processes. As teenagers and young adults have the highest risk of developing contact lens-related complications, this work highlights the importance of understanding ocular responses to contact lens wear across different ages.

2022-11-07 | PXD029188 | 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

Aspergillus flavus Induced Alterations in Tear Protein Profile Reveal Pathogen-Induced Host Response to Fungal Infection

Project description:Aspergillus flavus and Fusarium sp. are primary causative agents of keratitis that results in corneal tissue damage leading to vision loss. The tear proteome of control and keratitis patients was profiled and compared. A total of 1873 proteins from control and 1400 proteins from patient tear were identified by mass spectrometry. While 847 proteins were found to be glycosylated in the patient tear, only 726 were glycosylated in control tear. Some tear proteins showed alterations in their glycosylation pattern after infection. Complement system proteins, proteins specific for neutrophil extracellular traps and proteins involved in would healing were found only in the patient tear. The presence of these innate immune system proteins in patient's tear supports previous data indicating the involvement of neutrophil and complement pathways in antifungal defense. High levels of wound healing proteins in keratitis patient tear implied activation of tissue repair. The early appearance of host defense proteins and wound healing response indicates that tear proteins could be used as an early marker system for monitoring the progression of pathogenesis. Identification of negative regulators of the defense pathways in keratitis tear indicates an intricate balance of pro and anti-defense mechanisms operating in fungal infection of the eye.

2016-12-13 | PXD003825 | Pride

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