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Author Spotlight: Isolating Biomolecules from Mouse Tears — A Methodology for Molecular Analysis and Biomarker Research
Published on: July 19, 2024
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Experimental design considerations for studies of human tear proteins
Brooke M Harkness1, Deborah M Hegarty2, Julie A Saugstad3
1Casey Eye Institute, Oregon Health & Science University, Portland, OR, USA.
The Ocular Surface
|February 10, 2023
Summary
This study presents a reproducible workflow for analyzing individual human tear proteomes without pooling samples. This method allows for the discovery of protein biomarkers by accounting for factors influencing tear protein profiles.
Area of Science:
- Ophthalmology
- Proteomics
- Biomarker Discovery
Background:
- Human tears contain diverse proteins crucial for ocular surface health.
- Existing tear proteome analysis often requires pooling samples, limiting individual-factor studies.
- Reproducible methods are needed to analyze tear proteins, considering various influencing factors.
Purpose of the Study:
- To develop and evaluate a workflow for proteomic analysis of individual human tear samples.
- To enable biomarker discovery by analyzing tear proteins without sample pooling.
- To identify factors that influence tear protein profiles in clinical samples.
Main Methods:
- Tears collected via Schirmer strip, individually stored, and processed for proteomics.
- Protein extraction, digestion (S-Trap), and labeling (TMT) followed by 2D chromatography and mass spectrometry.
- Internal reference scaling (IRS) used for comparing TMT experiments.
Main Results:
- Demonstrated successful proteomic analysis of individual tear samples without pooling.
- Identified variations in tear protein subsets influenced by geographic collection site, contact lens use, and tear volume.
Conclusions:
- Proteomic analysis of individual human tear samples is feasible without pooling.
- Analytical workflows must account for confounding factors like collection site and contact lens use.
- This approach enhances opportunities for protein biomarker discovery in diverse clinical populations.
