Analysis of tear proteins by one- and two-dimensional thin-layer isoelectric focusing, sodium dodecyl sulfate electrophoresis and lectin blotting. Detection of a new component: cystatin C

Background: Isoelectric focusing (IEF) of tear proteins has not yet been ca rried out in a satisfactory way. Two-dimensional (2D) electrophoresis, espe cially in the combination of IEF with SDS, is able to differentiate between proteins in detail. The purpose of this study was therefore to analyze tea r proteins by 1D IEF alone and in combination with a 2D pattern, and by IEF followed by lectin staining. Methods: Ampholines, covering a broad range f rom pH 3 to pH 10, were applied. After IEF, semi-dry blotting and incubatio n with a group II lectin and two group V lectins was performed. Results: Te ar proteins could be separated into 31 single bands. Tear-specific pre-albu min (TSPA), lactoferrin, sIgA, IgG and lysozyme were found to be main compo nents. Isoelectric points (IEPs, pIs) of all proteins separated were determ ined by comparison with IEF standards. 2D patterns of IEF and SDS electroph oresis were obtained for the main subunit components of lactoferrin, sIEA, TSPA, and lysozyme. An additional new component of considerable concentrati on was focused at pI 8.6 with a subunit MW of 14 kDa. With s-WGA a componen t at an IEP of 5.2 was visualized, representing transferrin. With SNA, lact oferrin stained as a sharp main band at pi 5.1 with three additional weaker bands at IEPs from 4.8 to 4.9. At IEPs between 4.4 and 6.1, multiple compo nents of sIgA were stained with MAA. The sugar specificity of transferrin a t pI 5.2 was beta-GlcNAc. Lactoferrin showed glycation with NANA-alpha-2-6- Gal or NANA-alpha-2-6-GalNAc, whereas the sugar specificity of sIgA was NAN A-alpha-2-3-Gal. Conclusions: The investigative strategy applied here, incl uding IEF alone, in combination with SDS-electrophoresis, and SDS-electroph oresis followed by lectin staining proved to be a reproducible method for t ear protein analysis of hitherto unexperienced capacity. Lectin-stained ban ds of native tear proteins are not uniformly glycated by one sugar residue, but show various sugar specificities. IgA as a whole molecule is specifica lly glycated with NANA-alpha-2-3-Gal.