Purchase this article with an account.
P. Ramamoorthy, M. Thangavelu, H. L. Chandler, J. J. Nichols; Optimization of Methods for Analysis of High Molecular Weight Tear Glycoproteins and Tear Mucins. Invest. Ophthalmol. Vis. Sci. 2010;51(13):4188.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
To optimize tear collection methods, gel electrophoretic techniques and mass spectrometric analysis associated with analysis of high molecular weight (HMW) tear glycoproteins and tear mucins.
Tear samples were collected by microcapillary tube extraction from the inferior tear meniscus and by a wash method involving saline irrigation of the ocular surface. Samples were obtained from both eyes of 20 subjects including 10 normal non-lens wearers and 10 asymptomatic hydrogel contact lens wearers. All samples were stored at -80°C until analysis. Samples were subjected to Bradford assay, 3-8% tris acetate gel electrophoresis at 150 V for 1.5 hours, followed by periodic acid Schiff (PAS) staining, Coomassie staining, band densitometry and liquid chromatography-mass spectrometry (LC-MS) analysis. Human NCBI and an exclusive mucins database were searched. Western blotting for MUC5AC (antibody 1-13M1) and MUC4 (antibody 1G8) were performed to verify presence of tear mucins. Appropriate non-parametric tests were used to assess differences.
The average age of subjects was 30.8 ± 7.61 and 50% were female. Average protein concentration by Bradford assay was 0.61 ± 0.8 µg/µl for the microcapillary method and 1.03 ± 0.19 µg/µl for the wash method (p<0.0001). Protein bands were observed by Coomassie staining at ~ 500, 180, 97, 66, 55 and 40 kDa. The HMW 500 kDa band was significantly more dense for the wash samples compared to tear samples (p = 0.002). Values for the normal non lens wearers and contact lens wearers were not significantly different. LC-MS analysis of the 500 kDa tear bands of both subject groups identified several glycoproteins including heparin sulfate proteoglycan perlecan, glycoprotein 340, secretory IgA receptor and MUC4 (Table 1). Tear and wash samples were positive for MUC5AC and MUC4 in the western blots.
Tear protein concentration is greater in the eye wash sample compared to microcapillary extraction. Tris-acetate gel electrophoresis and LC-MS offer a novel approach to assess HMW tear proteins and mucins.
This PDF is available to Subscribers Only