Two-dimensional analysis of tear protein patterns of diabetic patients

Abstract

In diabetic patients, dry eye and other ocular surface diseases occur more often than in healthy subjects. The aim of this study was to analyze the tear protein patterns of patients suffering from diabetes mellitus type II (dia) and to compare them to the patterns of healthy volunteers (ctrl). Tear proteins of nonstimulated tears of 20 patients (ctrl n=10, dia n=10) were separated using two-dimensional electrophoretic techniques. The protein patterns of each group were analyzed by a multivariate analysis of discriminance. Furthermore, for all spots of each gel, a 50 x 50 variables pH/Mr (molecular weight) array was generated and subsequently analyzed by a multivariate analysis of discriminance. Additionally, an artificial neural network was trained using the matrix data as input and a sensitivity analysis was performed to figure out, which spots were the most important to differentiate between the tear protein patterns. In both groups a complex staining pattern could be obtained. In diabetic patients significantly more spots were detected compared to the control group (P<0.02). The analysis of discriminance found a highly significant difference between dia and ctrl (P<0.00001). Using the matrix data, the analysis of discriminance showed a significant difference between the two groups, too (P<0.0001). The sensitivity analysis by means of the artificial neural network revealed several spots that were more expressed or more frequently present in the diabetic group. Our findings reveal that the composition of tear proteins of diabetic patients is different from that of healthy subjects. The use of the two-dimensional electrophoretic technique could give more insight into the diabetic-related changes in the tear film composition.

Optical quality of the diabetic eye: a review

“…25,26 This could be the result of glycosylation of tear proteins that often leads to changes in the protein structure that may influence protein function. 25 Moreover, Grus et al 26 observed differences in protein patterns in the molecular weight range of 30-50 kDa, although they could not identify one single peak that was present in all diabetic patients. These proteins were not observed in healthy subjects and could be decisive therefore in the pathogenesis of DM and/or the development of eye-related complications of diabetic disease.…”

“…[16][17][18][19][20][21][22][23][24][25][26] Some ocular manifestations of DM are associated with lachrymal gland dysfunction and have been related to dry eye. Several clinical studies have demonstrated that people with DM are more vulnerable to dry eye than healthy subjects.…”

“…86,87 Even though there are no studies evaluating the impact that these factors could have on HOAs in people with DM, these do have a thicker and more convex lens, [54][55][56] can develop premature cataracts, [9][10][11] and suffer a series of changes associated with lachrymal gland dysfunction and dry eye syndrome. [16][17][18][19][20][21][22][23][24][25][26] Patients with DM who undergo laser-assisted in situ keratomileusis are also at a significantly higher risk of developing postoperative epithelial complications, and refractive results tend to be worse compared with healthy people. 88 Few authors have investigated HOAs in people with DM.…”

Diabetic retinopathy: Proteomic approaches to help the differential diagnosis and to understand the underlying molecular mechanisms

“…Electrophoresis [86], protein arrays [87] and various mass spectrometry-related techniques [85,88] have been applied. Various forms of chemical labeling [39,43], SRM/MRM-based targeted proteomics and label free quantification have been used for tear studies but no PRM or SWATH analyses have been carried out so far [89,90].…”

Proteomics in ocular fluids

“…Tears can be eluted from these strips using buffers compatible for further analysis [18]. The minimal invasiveness of the methods of sample collection makes tears a very interesting option for proteomic studies, not only in ocular diseases, but also in other conditions, e.g., diabetes mellitus [19][20][21][22] or cancer [23]. Tear film proteins were extensively analyzed by HPLC [24,25], 2-DE [26,27] and other chromatographic approaches [28].…”