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.

MicroRNAs are non-coding RNAs that serve as regulatory molecules in a variety of pathways such as inflammation, metabolism, homeostasis, cell machinery, and development. With the progression of sequencing methods and modern bioinformatics tools, novel roles of microRNAs in regulatory mechanisms and pathophysiological states continue to expand. Advances in detection methods have further enabled larger adoption of studies utilizing minimal sample volumes, allowing the analysis of microRNAs in low-volume biofluids, such as the aqueous humor and tear fluid. The reported abundance of extracellular microRNAs in these biofluids has prompted studies to explore their biomarker potential. This review compiles the current literature reporting microRNAs in human tear fluid and their association with ocular diseases including dry eye disease, Sjögren’s syndrome, keratitis, vernal keratoconjunctivitis, glaucoma, diabetic macular edema, and diabetic retinopathy, as well as non-ocular diseases, including Alzheimer’s and breast cancer. We also summarize the known roles of these microRNAs and shed light on the future progression of this field.

This study discovers the complement protein profile in the aqueous humor (AH) of human subjects and investigates its association with primary open-angle glaucoma (POAG) pathogenesis. Among the 32 complement proteins identified, 22 were highly abundant and detected in more than 50% of AH samples. The most predominant active complement proteins in the AH are C3, C4B, C4A, CFB, CFD, and C9. Additionally, the most prevalent complement regulators and receptors include CLU, SERPING1, F2, CFH, CFI, and VTN. Significant alterations in complement proteins were observed in individuals with POAG compared to those with cataracts. Specifically, complement protein F2 was upregulated, while C8G, C6, and CFH were downregulated in POAG samples. Stratification of the samples by race and sex revealed distinct alterations of complement proteins in patients with POAG. In the African American cohort, five complement proteins (C4A, C4B, F2, C7, and C3) were upregulated in POAG compared to cataract patients. In the Caucasian cohort, eight complement proteins (C3, SERPING1, CFI, CLU, CFHR1, C8G, C6, and CFH) were downregulated in the POAG samples compared to the cataract samples. Within the male cohort, three complement proteins (CLU, C6, and CFH) were downregulated in POAG patients compared to those with cataracts. Whereas, within the female cohort, two complement proteins (C4B and F2) were upregulated and one (C8G) downregulated in the POAG samples when compared to cataracts. Discerning these changes in the AH complement protein profile will assist in the development of tailored therapies to modulate the complement system for managing ocular disorders. These insights may also lead to novel biomarkers for diagnosing and monitoring disease progression.

To generate a comprehensive profile of microRNAs (miRNAs) present in human tear fluid using next-generation sequencing (NGS), establish a reference miRNome for healthy human tear fluid, and investigate whether miRNA expression varies by sex, race, or age. Tear samples were collected from 32 adults using Schirmer strips. RNA was isolated using the miRNeasy Serum/Plasma Kit, and cDNA libraries were prepared using the QIAseq miRNA Library Kit. Barcoded libraries were sequenced on the NovaSeq 6000 platform. Bioinformatic analyses included adapter trimming, alignment to miRBase, normalization with DESeq2, and functional annotation using multiMiR and clusterProfiler. Differential expression analysis was performed to evaluate variability across demographic groups. We identified 318 miRNAs constitutively expressed in tear fluid, 246 of which were detected in all subjects. The most abundant miRNAs included hsa-let-7a-5p, hsa-let-7f-5p, hsa-let-7b-5p, hsa-let-7g-5p, and hsa-let-7c-5p. Prominent miRNA families included let-7 (13 members), miR-30 (10), miR-8 (10), miR-10 (8), and miR-17 (8). Functional enrichment analyses indicated that tear miRNAs may regulate pathways involved in transcriptional control, cytoskeletal organization, MAPK signaling, and neurodegenerative processes. Samples processed in two batches several months apart showed strong concordance, confirming methodological reproducibility. Two miRNAs, hsa-miR-375-3p and hsa-miR-708-5p, were significantly increased in White subjects, whereas hsa-miR-106b-3p and hsa-miR-205-3p showed a nonsignificant increase in females. This study defines a high-resolution reference profile of the human tear fluid miRNome, demonstrating its stability, diversity, and functional relevance. These data establish a robust baseline for future studies investigating disease-associated alterations in tear miRNAs. Observed demographic associations remain exploratory and require larger, adequately powered cohorts.