Tear Proteomics in Keratoconus: A Quantitative SWATH-MS Analysis

Abstract

Purpose To elucidate dysregulated proteins in keratoconus (KC) to provide a better understanding of the molecular mechanisms that lead to the development of the disease using sequential window acquisition of all theoretical mass spectra (SWATH-MS) as a protein quantification tool of the tear proteomic profile. Methods Prospective cross-sectional study that includes 25 keratoconic eyes and 25 healthy eyes. All participants underwent a clinical, tomographic, and aberrometric exam. Tear sample was collected using Schirmer strips and analyzed by liquid chromatography with tandem mass spectrometry. SWATH-MS was used as a quantification tool of the tear proteomic profile. The expression of the quantified proteins was compared between groups, and the biological and molecular functions of the dysregulated proteins as well as their functional relationships were studied by in silico analysis. Results A total of 203 proteins were quantified in tear samples of patients with KC and control participants, of which 18 showed differential expression between groups ( P < 0.05). An increase in the expression of 7 proteins and a decrease in the expression of 11 proteins were observed. Protein–protein interactions and gene ontology analysis showed the involvement of these dysregulated proteins in structural, inflammatory-immune, iron homeostasis, oxidative stress, and extracellular matrix proteolysis processes. Conclusions Tear protein quantification has revealed the dysregulation of proteins involved in biological processes previously associated with KC. Among them, iron homeostasis should be highlighted as a relevant pathway in the KC pathophysiology, and it should be taken into account in the development of therapeutic targets to cope with tissue damage derived from iron accumulation and toxicity.

Nutritional and Metabolic Imbalance in Keratoconus

“…Due to Fe peripheral deposition, it was hypothesized that central unavailability of Fe contributes to KC development, because it works as a cofactor of lysyl hydroxylase (LH), which is required for the collagen crosslinking process. While no significant differences were found in the serum levels of Fe [59], KC patients show lower levels of iron-binding proteins serotransferrin and lactoferrin in tears [47,68] and the corneal epithelium [69], suggesting that iron homeostasis is locally altered in KC cornea. Interestingly, certain polymorphisms of the transferrin gene have been identified as risk factors for KC [70].…”

“…The hypothesis that Vit D deficiency is common in KC patients was confirmed years later when it was shown that KC patients displayed lower serum levels of Vit D when compared to age-matched controls [44][45][46]. Interestingly, KC patients exhibit downregulation of proteins involved in Vit D transport (e.g., Vit Dbinding protein) and activation [47]. Moreover, an association between Vit D deficiency and KC prevalence was suggested in people living in Middle East regions [46].…”

Alzheimer’s Disease Seen through the Eye: Ocular Alterations and Neurodegeneration

“…Hopefully, these new tools will help to overcome the limitations related to the small volume of tear samples. To date, there are already numerous ocular (e.g., keratoconus, dry eye, and glaucoma) and systemic conditions (such as diabetes mellitus, thyroid dysfunction, and neurological diseases) that can be detected and evaluated by biomarkers at the lacrimal level [ 125 , 126 ].…”

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

“…Recently, our group carried out a quantification approach of the tear proteome of patients with KC and control participants, using for the first time the sequential window acquisition for all theoretical mass spectra to identify the main dysregulated proteins in the tears of patients with KC. 22 In this way, results from the quantitative study revealed that dysregulated proteins were involved in the central processes of the KC pathophysiology, such as inflammation, oxidative stress, matrix proteolysis, and iron homeostasis. 22 However, the qualitative approach followed in this study aims to complement our previous results, trying to overcome the main handicap of quantitative studies (in which it is necessary to recognize a critical number of peptides to achieve protein quantification), and to obtain an even more extensive view of the tear proteomic profile of the patients with KC.…”

“… 22 In this way, results from the quantitative study revealed that dysregulated proteins were involved in the central processes of the KC pathophysiology, such as inflammation, oxidative stress, matrix proteolysis, and iron homeostasis. 22 However, the qualitative approach followed in this study aims to complement our previous results, trying to overcome the main handicap of quantitative studies (in which it is necessary to recognize a critical number of peptides to achieve protein quantification), and to obtain an even more extensive view of the tear proteomic profile of the patients with KC. Based on these factors, we performed a shotgun analysis to massively identify the tear proteome of patients with KC, which could highlight proteins undetected in the quantitative analysis and enlarge our knowledge of the disease's pathophysiology.…”