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Mass spectrometry protocol for tear fluid proteome profiling and biomarker discovery

ABSTRACT: Protocol for tear fluid proteomics using Schirmer strips and LC-MS/MS.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Petra Hamerlik

LAB HEAD: Petra Hamerlik

PROVIDER: PXD070688 | Pride | 2026-01-12

REPOSITORIES: Pride

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			Action	DRS
	20240608-0547_EXP1_003782_070624_SL_TEARS_STAR_1000ng_Sample_1.raw	Raw
	20240608-0958_EXP1_003783_070624_SL_TEARS_STAR_1000ng_Sample_2.raw	Raw
	20240608-1410_EXP1_003784_070624_SL_TEARS_STAR_1000ng_Sample_3.raw	Raw
	20240608-1823_EXP1_003785_070624_SL_TEARS_STAR_1000ng_Sample_4.raw	Raw
	20240608-2235_EXP1_003786_070624_SL_TEARS_STAR_1000ng_Sample_5.raw	Raw

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Mass spectrometry protocol for tear fluid proteome profiling and biomarker discovery.

Juul-Elbaek Kirstine K Peiris-Pagès Maria M Akimov Vyacheslav V Rebollido-Pedrido Irene I Reardon Mark M Blagoev Blagoy B Hamerlik Petra P

STAR protocols 20251215 1

Here, we present a protocol for tear fluid proteomics using Schirmer strips and liquid chromatography-tandem mass spectrometry (LC-MS/MS). It includes tear sampling from human individuals and mice, followed by protein digestion, peptide desalting, and purification via solid-phase extraction. Supporting both data-dependent acquisition (DDA) and data-independent acquisition (DIA), this 5-h workflow maximizes protein recovery, minimizes contaminants, and enables reproducible biomarker discovery for ...[more]

PMID: 41401067

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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

Dog tear film proteome in-depth analysis

Project description:In this study, mass spectrometry was used to explore the canine tear proteome. Tear samples were obtained from six healthy dogs, and one-dimensional sodium dodecyl sulphate polyacrylamide gel electrophoresis (1D SDS-PAGE) was used as a first step to separate intact proteins into 17 bands. Each fraction was then trypsin digested and analysed by matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry (MALDI-TOF-MS/MS) to characterize the protein components in each fraction. In total, 125 tear proteins were identified, with MCA (Major Canine Allergen), Serum albumin, UPF0557 protein C10orf119 homolog, Collagen alpha-2(I) chain, Tyrosine -protein kinase Fer, Keratine type II cytoskeletal, Beta-crystallin B2, Interleukin-6 and Desmin occuring as the most confident ones with the highest scores. The results showed that the proteomic strategy used in this study was successful in the analysis of the dog tear proteome. To the best of our knowledge, this study is the first to report the comprehensive proteome profile of tears from healthy dogs by 1D SDS PAGE and MALDI-TOF.

2015-11-23 | PXD003124 | Pride

Precision in Tear Fluid Biomarker Discovery: Quantitative Proteomic Profiling of Small-Volume, Individual Samples Using Capillary Tube Collection

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

Rapid Tear Proteomics via In-Capillary Digestion for Biomarker Discovery

Project description:Tear fluid is emerging as a valuable resource for biomarker discovery, but its low sample volume and dynamic composition pose significant challenges for analytical testing. Most tear proteomics studies have focused on samples collected with Schirmer strips, while capillary tube collection has received less attention. To address these challenges, we developed a novel in-capillary trypsin digestion workflow that requires as little as 0.5 μL of tear fluid for bottom-up shotgun proteomics. This method uses liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify a higher number of proteins with enhanced efficiency compared to previously reported methods, which typically required 5-10 μL of pooled tear fluid. Using the same microcentrifuge tube for both tear collection and sample processing, this streamlined workflow simplifies sample handling and minimizes both sample loss and experimental errors associated with sample transfer. This method also efficiently reduced sample processing time to under 2 hours prior to overnight trypsin di-gestion, compared to the 5-8 hours required by other methods. With this workflow, we identified 500-800 proteins per 0.5 μL sample without the need for fractionation, allowing for at least three technical replicates. This optimized workflow greatly enhances the efficiency of tear proteomics for biomarker discovery.

2025-05-07 | PXD056978 | Pride

Mass spectrometric detection of keratins in tear fluid

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.

2025-03-03 | PXD061409 | Pride

Potential tear biomarkers for the early diagnosis of Parkinson's disease- A pilot study

Project description:Parkinson's disease (PD) is the second most common neurodegenerative disease after Alzheimer's disease. The purpose of this study was to analyze the profile of the tear proteome of patients with idiopathic PD (iPD), carriers of the E46K-SNCA mutation and healthy subjects (CT) and to identify biomarkers for the early diagnosis of PD. An observational, prospective and case-control pilot study was carried out in 24 patients with iPD, 3 carriers of the E46K-SNCA mutation and 27 CT subjects. The patients' neurological involvement was scored and their tear samples were analyzed using nano-liquid chromatography-mass spectrometry (nLC-MS/MS). These analyses led to the identification of 560 tear proteins in which some of the deregulated proteins were involved in immune response, apoptosis, collagen degradation, protein synthesis, defense and altered lysosomal function. Of these proteins, six related to neurodegenerative processes, showed a good capacity to classify patients and controls. These findings revealed that some proteins were upregulated in the tears of PD patients, mainly those involved in lysosomal function. It is worth highlighting the importance of this study in identifying tear proteins implicated in neurodegeneration and its relationship with PD patients with an aggressive disease phenotype.

2022-02-14 | PXD028811 | Pride

SH2B1 Promotes Apoptosis in Diabetic Cataract via p38 MAPK Pathway

Project description:Diabetic patients face an increased risk of developing cataracts, with unclear mechanisms. Our study illuminates these mechanisms by identifying differentially expressed proteins in the lens anterior capsule of diabetic cataract (DC) and age-related cataract patients using quantitative proteomics. We found SH2B1 to be crucial in DC progression. Reduced SH2B1 expression was confirmed through PCR and Western blotting in patient samples, diet-induced obese mice, and high-glucose (HG)-cultured human lens epithelial cells. Under HG conditions, cell proliferation decreased, while migration and apoptosis, alongside changes in Bcl2 and caspase 3 expression, increased. Overexpressing SH2B1 alleviated these changes and influenced the p38 MAPK signaling pathway. This suggests SH2B1 and the p38 MAPK pathway as significant in DC pathogenesis and potential therapeutic targets. Clinically, this could lead to therapies aimed at halting or slowing DC progression.

2025-05-07 | PXD059243 | 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 proteins altered in patients with persistent eye pain after refractive surgery: biomarker candidate discovery

Project description:Some patients develop persistent eye pain after refractive surgery, but the factors that cause or sustain pain are unknown. We tested whether tear proteins of patients with pain 3 months after surgery differ from patients without pain. Patients undergoing refractive surgery (LASIK or PRK) were recruited from 2 clinics and tears were collected 3 months after surgery. Participants rated their eye pain using a numerical rating scale (NRS, 0 – 10; no pain – worst pain) at baseline, 1 day, and 3 months after surgery. Using tandem mass tag (TMT) proteomic analysis, we examined tears from participants with NRS > 3 at 3 months (n=16) and a comparator group with NRS < 1 at 3 months after surgery (n=32). A subset of proteins (83 of 2748 detected, 3.0%) were associated with pain 3 months after surgery. High-dimensional statistical models showed that magnitude of differential expression was not the only important factor in classifying tear samples from pain patients. Models utilizing 3 or 4 proteins had better classification performance than single proteins and represented differences in both directions (higher or lower in pain). Thus, patterns of protein differences may serve as biomarkers of post-surgical eye pain as well as potential therapeutic targets.

2025-05-25 | PXD051071 | Pride

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