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

## TL;DR

This paper introduces a 5-hour protocol for analyzing tear fluid proteins using mass spectrometry to discover disease biomarkers in a non-invasive way.

## Contribution

A standardized, efficient tear proteomics workflow using Schirmer strips and LC-MS/MS for biomarker discovery in multiple diseases.

## Key findings

- The protocol enables high protein recovery and low contamination in tear fluid samples.
- It supports both DDA and DIA mass spectrometry methods for reproducible biomarker discovery.
- Tear fluid is shown to be a viable and non-invasive source for clinical proteomics.

## Abstract

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 ocular, neurodegenerative, and systemic diseases. Tear fluid’s non-invasive collection and rich proteome make it ideal for clinical proteomics and personalized medicine.

•Guidance for aseptic tear fluid collection using Schirmer strips in humans and mice•Steps for protein extraction and digestion using GuaHCl, LysC, and trypsin•Instructions for peptide desalting with SOLA plates for LC-MS/MS analysis•Procedures for minimizing variability in tear-based biomarker discovery

Guidance for aseptic tear fluid collection using Schirmer strips in humans and mice

Steps for protein extraction and digestion using GuaHCl, LysC, and trypsin

Instructions for peptide desalting with SOLA plates for LC-MS/MS analysis

Procedures for minimizing variability in tear-based biomarker discovery

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

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 ocular, neurodegenerative, and systemic diseases. Tear fluid’s non-invasive collection and rich proteome make it ideal for clinical proteomics and personalized medicine.