Quantitative Top-Down Proteomics of Human Tears Reveals Proteoform Changes Related to Age

Significance of the Topic

Human tear fluid is a minimally invasive source of protein biomarkers that reflect ocular and systemic health. Detailed proteoform profiling in tears can uncover subtle molecular changes associated with aging, disease, or environmental stress, providing new insights into eye physiology and potential diagnostic markers.

Study Objectives and Overview

This study aimed to apply a novel label-free top-down quantitation workflow to detect age-related proteoform changes in human tear fluid. Two donor groups (young and old) were compared to identify differentially expressed intact proteoforms and assess the advantages of top-down analysis over traditional peptide-based methods.

Methodology and Instrumentation

Tear samples were collected noninvasively using Schirmer strips and extracted in aqueous buffer. After storage at low temperature, samples were thawed, centrifuged, and analyzed by liquid chromatography–mass spectrometry. Ultraviolet photodissociation (UVPD) at 213 nm on a modified Thermo Scientific Orbitrap Fusion Tribrid enabled comprehensive fragmentation of intact proteoforms. Data acquisition was performed at 120,000 resolution (m/z 200).

Data Analysis

Raw MS/MS data were processed in Proteome Discoverer with ProSightPD 4.0 (beta) nodes. A sliding window deconvolution algorithm improved signal-to-noise for coeluting charged species, and ProSight search workflows identified and quantified proteoforms directly at the intact mass level.

Main Results and Discussion

UVPD outperformed higher-energy collisional dissociation (HCD) and electron transfer dissociation (ETD) in fragmenting disulfide-rich regions, such as truncated lysozyme variants. Many detected proteoforms, particularly truncated forms of Proline Rich Protein 4 (PRP4), were not annotated in UniProt. Quantitative comparison revealed that certain PRP4 truncations were more abundant in young donors, while alternative truncated forms increased with age. Protein–protein association network analysis clustered PRP4 with other tear proteins (LACT, CST4) and central nervous system regulators, suggesting functional relevance. These age-dependent proteoform dynamics would remain hidden in peptide-centric workflows.

Benefits and Practical Applications

• Direct proteoform quantitation bypasses peptide inference, enhancing molecular specificity.
• UVPD enables sequencing across intact disulfide bonds and proline-rich regions.
• Label-free top-down workflows reduce sample preparation complexity and avoid labeling costs.
• Age-related proteoform biomarkers in tears may support noninvasive diagnostics and personalized monitoring of ocular health.

Future Trends and Potential Applications

• Integration of advanced fragmentation methods (UVPD, EThcD) for broader proteoform coverage.
• Automated top-down pipelines and real-time data analysis for clinical laboratories.
• Expansion to other biofluids (saliva, cerebrospinal fluid) for multi-organ biomarker discovery.
• Combining proteoform data with genomics and metabolomics in systems-level studies.

Conclusion

The implementation of label-free top-down proteomics with UVPD and ProSightPD 4.0 in human tear fluid permits high-resolution, proteoform-level quantitation. This approach reveals age-dependent molecular changes obscured by conventional peptide workflows and opens new avenues for noninvasive biomarker discovery in ocular and systemic health.

References

1. Von Thun und Hohenstein-Blaul N., Funke S., Grus F.H. Tears as a source of biomarkers for ocular and systemic diseases. Experimental Eye Research. 117:126–137 (2013).
2. Moehring F., Waas M., Keppel T.R., Rathore D., Cowie A., Stucky C., Gundry R.L. Quantitative top-down mass spectrometry identifies proteoforms differentially released during mechanical stimulation of mouse skin. Journal of Proteome Research. 8(6):2635–2648 (2018).