Shotgun Proteomics for the Identification and Profiling of the Tear Proteome of Keratoconus Patients

Abstract

Purpose The qualitative approach followed in this study aims to obtain an extensive view of the keratoconus (KC) tear proteome, which could highlight proteins previously undetected and enlarge our knowledge of the disease's pathophysiology. Methods Twenty-five patients diagnosed with KC and 25 control subjects were studied in a prospective, cross-sectional study. KC screening examinations, including clinical and tomographic examinations, were performed on all participants. Tear samples were collected using Schirmer strips and analyzed by liquid chromatography-tandem mass spectrometry in a data-dependent workflow. A spectral count was used as a semiquantification tool. The tear proteomes of both groups were identified and profiled, and the functional interactions and biological characterization of differential proteins were analyzed using in silico tools. Results We identified a total of 232 proteins, of whom 133 were expressed in both groups’ samples; 41 were observed only in control samples and 58 were identified just in tears of patients with KC. A semiquantitative analysis showed the dysregulation of 17 proteins in the KC samples. An in silico analysis linked proteins only expressed in KC samples to oxidative stress, skin development, and apoptosis. The dysregulation of proteins involved in iron transport, inflammation, oxidative stress, and protease inhibition was observed in the semiquantitative results. Conclusions A shotgun analysis showed that the tear proteome of patients with KC differed from controls by more than one-third of the total proteins identified, highlighting the relationship of the proteins only expressed in KC tears with processes of cell death, oxidative damage, and inflammation. The underexpression of proteins involved in iron pathways might support the iron imbalance as a contributing factor to cellular damage and death in KC disease.

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“…Historically, lactoferrin (LF), IgE and MMP-9 have been the most common translational biomarkers studied in tear film and their usefulness has been validated in dry eye disease [ 119 ], allergic conjunctivitis [ 120 ], keratoconus [ 121 ] and inflammatory conjunctivitis [ 122 , 123 ]. As for nAMD, the use of two-dimensional electrophoresis followed by the MALDI-TOF/TOF mass spectrometry approach in a recent study [ 67 ] has led to the identification of a set of dysregulated tear film proteins including several proteins related to inflammation and neovascularization, such as allograft inflammatory factor 1 (AIF1), ATP-dependent translocase (ABCB1) and annexin-1.…”

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“…Numerous research studies have recognized the role of oxidative stress, innate immunity, and inflammation pathways in the KC pathogenesis [ 125 , 126 ]. Higher levels of damaging free radicals and lower levels of antioxidant molecules were reported in KC samples [ 127 ].…”

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“…Alzheimer's dementia, Parkinson's disease, multiple sclerosis, breast cancer) 126 , 146 . Tear proteome profiling in keratoconus patients identified 58 proteins related to process of cell death, oxidative damage and inflammation specific to tears of patients with keratoconus; 41 proteins involved in iron pathways was found to be underexpressed in keratoconus patients and identified in control samples only 147 . Besides improving the knowledge of the disease's pathophysiology, Goni et al also analysed the tear proteome profile before and after treatments and identified biomarkers, mainly A-kinase anchor protein 13 (AKAP-13), altered by surgical treatment 148 .…”