Statistical Analysis The average protein and peptide counts per sample identified in all four workflows were compared via two-way ANOVA, and subsequent pairwise comparisons were performed using Tukeys multiple comparison test in order to control the family wise error rate [70]

Statistical Analysis The average protein and peptide counts per sample identified in all four workflows were compared via two-way ANOVA, and subsequent pairwise comparisons were performed using Tukeys multiple comparison test in order to control the family wise error rate [70]. 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. = 11), and each strip was cut longitudinally into two equal parts. In Method A, proteins were first extracted, and Schirmer strips were removed by filter-aided centrifugation prior to digestion. In Method B, Schirmer strips were cut into 5 mm pieces, and in-strip protein digestion was performed. Digested products from each method then underwent LCCMS/MS analysis using both collision-induced dissociation (CID) and higher-energy collisional dissociation (HCD) fragmentation techniques. The average number of unique proteins (= 11/group; * = 182; medium, = 147; low, = 373; and rare, = 2668). Since this workflow identified the greatest number of unique proteins, these 3370 unique proteins were further examined using bioinformatics approaches to determine the characteristics of the proteome of human tear film. The 50 most abundant proteins detected using in-strip protein digestion and HCD fragmentation are VP3.15 listed in Table 3. Open in a separate window Figure 3 Distribution of mean protein expression by sample proportion in the four different workflows. PeptideCspectrum matches VP3.15 (PSMs) from the 11 tear samples were log2 transformed for each digestion and fragmentation method performed to compare differences in mean protein expression between workflows. Further, proteins detected in the 11 samples VP3.15 were proportionally assessed and subdivided into four categories based on trends of detection for each method: High (shown in black; detected in 75% of samples), Medium (shown in green; detected in 50C75%), Low (shown in red; detected in 25C50%), and Rare (shown in blue; detected in 5C25%). Table 2 Total number of unique proteins identified in tear samples using four different workflows. and and functions to reverse cholesterol transport, preventing the build-up of free EZH2 cholesterol that can otherwise lead VP3.15 to meibomian gland dysfunction (MGD) [53]. Also, a previous study has shown that is upregulated in the tears of patients with diabetes [54]. Structural proteins of the ocular surface, such as keratins and myosins, are commonly found in tear fluid in both healthy and diseased conditions [55]. Keratins are produced by the meibomian gland and form a protective covering for the eye [56]. Myosins are ubiquitously expressed across cell types, and their cellular functions are diverse, including roles in polarization, movement, and exocytosis [57,58,59]. Specifically, myosin 6, found in highest abundance, is necessary for iris development [60]. In our analysis of human tear fluid with the chosen workflow, we were able to detect 26 keratin-family proteins and 15 myosin-family proteins. Consistent with other studies, lactotransferrin (and has been shown to negatively correlate with fluorescein tear break-up time (FTBUT), suggesting these proteins may play a role in dry eye symptoms [66]. levels have been shown to increase in tears in response to successful glaucoma treatment [66]. Since the tear film is an acellular biofluid, the majority of its protein and lipid contents are secreted by glandular and epithelial cells via exosomes. In our study, extracellular exosome and extracellular vesicle were highly enriched cellular components associated with tear proteins. Four major canonical pathways associated with the identified tear proteins were acute phase response signaling, glucocorticoid receptor signaling, liver X receptor/retinoid X receptor (LXR/RXR) signaling, and phagosome formation. The concentration of acute phase proteins (APPs) has been reported to change in response to inflammation [67]. Furthermore, the APP signaling pathway in the tear film is responsible for initiating immune cascades in the VP3.15 presence of inflammation, thus defining its role in the health of the ocular surface. A previous study found that changes in the tear fluid LXR/RXR signaling pathway are.