Proteomic Analysis of Tear Film Obtained from Diabetic Dogs

Abstract

Canine diabetes mellitus is a significant health burden, followed with numerous systemic complications, including diabetic cataracts and retinopathy, leading to blindness. Diabetes should be considered as a disease damaging all the body organs, including gastrointestinal tract, through a complex combination of vascular and metabolic pathologies, leading to impaired gut function. Tear film can be obtained in a non-invasive way, which makes it a feasible biomarker source. In this study we compared proteomic changes ongoing in tear film of diabetic dogs. The study group consisted of 15 diabetic dogs, and 13 dogs served as a control group. After obtaining tear film with Schirmer strips, we performed 2-dimensional electrophoresis, followed by Delta2D software analysis, which allowed to select statistically significant differentially expressed proteins. After their identification with MALDI-TOF (matrix assisted laser desorption and ionisation time of flight) spectrometry we found one up-regulated protein in tear film of diabetic dogs—SRC kinase signaling inhibitor 1 (SRCIN1). Eight proteins were down-regulated: phosphatidylinositol-4 kinase type 2 alpha (PI4KIIα), Pro-melanin concentrating hormone (Pro-MCH), Flotillin-1, Protein mono-ADP ribosyltransferase, GRIP and coiled coil domain containing protein 2, tetratricopeptide repeat protein 36, serpin, and Prelamin A/C. Identified proteins were analyzed by Panther Gene Ontology software, and their possible connections with diabetic etiopathology were discussed. We believe that this is the first study to target tear film proteome in canine diabetes. We believe that combined with traditional examination, the tear film proteomic analysis can be a new source of biomarkers both for clinical practice, and experimental research.

Procoagulant and anticoagulant plasma indicators in diabetic dogs showing increased antithrombin III levels in canine diabetes mellitus

“…Blood was collected in EDTA and non-EDTA tubes from each dog in the sitting position from the cephalic vein using a vacuum system. All the following procedures were carried on as in our previous experiment [ 38 ]. In detail, before sampling, the skin was shaved, and the whole procedure was carried out under sterile conditions.…”

Changes in Tear Proteomic Profile in Ocular Diseases

“…For protein extraction, our team used 8 M of urea buffer containing dithiothreitol and CHAPS in the presence of protease inhibitor cocktail. The process lasted for 3 h in 4 °C and was ended by centrifuging (5000× g for 20 min) [ 23 , 24 , 25 ]. Moreover, Ihnatko chose the addition of urea in solubilization buffer for protein extraction [ 26 ].…”

“…NanoDrop was used to determine protein level in studies correlated with age-related changes to human tear composition [ 33 ] and with a prediction of vernal keratoconjunctivitis reactivation [ 34 ]. MaestroNano was applied by our team for the protein measurements of tear fluid in macular edema, due to the neovascular age-related macular degeneration and analysis of tear film obtained from diabetic dogs [ 24 , 25 ]. In terms of protein concentration determination, infrared electromagnetic radiation is also suitable.…”

“…Protein and peptides concentration and purification can be also performed by ZipTip with a 0.6 or 0.2 µL bed of chromatography media concentrating and purifying femtomoles to the picomoles of protein samples. These chromatographic micro-beds are suitable for sensitive analyses such as MALDI-TOF MS [ 24 , 25 ]. Lipids can by removed in the route of double extraction by ice-cold chloroform:methanol mixture (1:1) at 1200 rpm [ 17 ].…”

“…Lipids can by removed in the route of double extraction by ice-cold chloroform:methanol mixture (1:1) at 1200 rpm [ 17 ]. Another solution for sample purification as well as precipitation is to apply cleanup kits, which use selective precipitation to remove ionic contaminants such as detergents, lipids and phenolic compounds, improving 2D electrophoresis resolution and reproducibility [ 24 , 25 ].…”