Comparison of Different Mass Spectrometry Workflows for the Proteomic Analysis of Tear Fluid

• Published in: International journal of molecular sciences Vol. 23; no. 4; p. 2307
• Main Authors: Jones, Garrett, Lee, Tae Jin, Glass, Joshua, Rountree, Grace, Ulrich, Lane, Estes, Amy, Sezer, Mary, Zhi, Wenbo, Sharma, Shruti, Sharma, Ashok
• Format: Journal Article
• Language: English
• Published: Switzerland MDPI AG 19.02.2022; MDPI
• Subjects: Adult; Biomarkers; Biomarkers - metabolism; Cornea; Disease; dry eye disease; Dry Eye Syndromes - metabolism; Eye diseases; Eye Proteins - metabolism; Female; Heat shock proteins; Humans; Immunoglobulins; Lipids; Male; Mass spectrometry; Mass Spectrometry - methods; Mass spectroscopy; Methods; Middle Aged; Multilayers; Nutrients; ocular surface; Ontology; Peptides; Proteins; Proteomics; Proteomics - methods; Scientific imaging; Spectroscopy; tear film; Tears - chemistry; Workflow; Young Adult; mass spectrometry; tear film; dry eye disease; proteomics; biomarkers; ocular surface
• ISSN: 1422-0067; 1661-6596; 1422-0067
• DOI: 10.3390/ijms23042307

The tear film is a multi-layer fluid that covers the corneal and conjunctival epithelia of the eye and provides lubrication, nutrients, and protection from the outside environment. Tear fluid contains a high concentration of proteins and has thus been recognized as a potential source of biomarkers for ocular disorders due to its proximity to disease sites on the ocular surface and the non-invasive nature of its collection. This is particularly true in the case of dry eye disease, which directly impacts the tear film and its components. Proteomic analysis of tear fluid is challenging mainly due to the wide dynamic range of proteins and the small sample volumes. However, recent advancements in mass spectrometry have revolutionized the field of proteomics enabling unprecedented depth, speed, and accuracy, even with small sample volumes. In this study using the Orbitrap Fusion Tribrid mass spectrometer, we compared four different mass spectrometry workflows for the proteomic analysis of tear fluid collected via Schirmer strips. We were able to establish a method of in-strip protein digestion that identified >3000 proteins in human tear samples from 11 healthy subjects. Our method offers a significant improvement in the number of proteins identified compared to previously reported methods without pooling samples.