Dataset Information

Evaluation of pre-processing methods for tear fluid proteomics using proximity extension assays.

ABSTRACT: Tear fluid forms a potential source for biomarker identification, and can be minimal invasively collected via Schirmer strips. The lack of knowledge on the processing of Schirmer strips however complicates the analysis and between-study comparisons. We studied two different pre-processing methods, specifically the use of punches of the strip versus elution of the strip in a buffer. Tear fluid filled Schirmer strips were collected from 5 healthy participants, and divided into two halves over the length of the strip. In either part, punches or eluates were obtained from 4 different locations, from the first part touching the eye (head) to the end, to assess the protein distribution along the strips. The levels of 92 inflammatory proteins were measured in the punches/eluates using proximity extension assays. The punch method yielded higher protein detectability compared to the elution method (76% vs 66%; p ≤ 0.001). Protein expression level was found to be slightly higher in the head of the strip, however, 3 out of 5 punches from the head failed quality control. Protein expression levels over the remaining parts of the strips were similar. Our study showed beneficial use of punches of any part of the strip except the head in future biomarker research.

SUBMITTER: Vergouwen DPC

PROVIDER: S-EPMC10023677 | biostudies-literature | 2023 Mar

REPOSITORIES: biostudies-literature

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Evaluation of pre-processing methods for tear fluid proteomics using proximity extension assays.

Vergouwen Daphne P C DPC Schotting Amber J AJ Endermann Tanja T van de Werken Harmen J G HJG Grashof Dwin G B DGB Arumugam Sinthuja S Nuijts Rudy M M A RMMA Ten Berge Josianne C JC Rothova Aniki A Schreurs Marco W J MWJ Gijs Marlies M

Scientific reports 20230317 1

Tear fluid forms a potential source for biomarker identification, and can be minimal invasively collected via Schirmer strips. The lack of knowledge on the processing of Schirmer strips however complicates the analysis and between-study comparisons. We studied two different pre-processing methods, specifically the use of punches of the strip versus elution of the strip in a buffer. Tear fluid filled Schirmer strips were collected from 5 healthy participants, and divided into two halves over the ...[more]

PMID: 36932139

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Project description:PurposeScleritis is an extremely painful and potentially blinding inflammation of the sclera with unknown pathogenesis and unpredictable course. To gain insight in its disease process and identify biomarker candidates, we performed extensive proteomics in serum and tear fluid.DesignProspective multicenter cohort study.ParticipantsA total of 121 patients with noninfectious scleritis (of which 39 active cases), 30 healthy controls, and 23 disease controls (uveitis and rheumatoid arthritis) were enrolled in the Netherlands from 2020 to 2022.MethodsSerum, tear fluid of both eyes, and clinical data were gathered. The level of 368 inflammatory proteins was measured using proximity extension assays. Results were validated in an independent cohort of 15 patients with scleritis, and using addressable laser bead immunoassay, or enzyme-linked immunoassays. In addition, we studied an extended panel of matrix metalloproteinases in tear fluid of necrotizing scleritis with addressable laser bead immunoassay.Main outcome measuresStatistically significant differences in the level of inflammatory proteins between patients with scleritis and control groups.ResultsProteomics revealed 18 significantly upregulated or downregulated serum proteins in active scleritis cases compared with all control groups in both the discovery cohort and the validation cohort. The most upregulated protein was nuclear migration protein nudC (NudC; P = 0.0032), a protein involved in neurogenesis. The other significant hits included proteins involved in T-cell activation, apoptosis, epithelial barrier maintenance, and angiogenesis. Our tear fluid analysis showed matrix metalloproteinase 9 (MMP9) to be upregulated in the tear fluid of patients with scleral necrosis.ConclusionsThe results of our proteomics analysis suggest a role for neurogenesis, T-cell activation, disruption of epithelial barrier, and angiogenesis in the pathogenesis of scleritis, and highlight MMP9 and NudC as biomarkers with potential clinical relevance.Funding disclosuresThe authors have no proprietary or commercial interest in any materials discussed in this article.

Dataset Information

Evaluation of pre-processing methods for tear fluid proteomics using proximity extension assays.

Publications

Evaluation of pre-processing methods for tear fluid proteomics using proximity extension assays.

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