Identification of human tear protein constituents by high resolution two-dimensional gel electrophoresis

Purpose: To separate and identify human tear proteins by two-dimensional (2D) gel eleclrophoresis, paying particular attention to isoforms present. Methods: Reflex tears were collected from 12 healthy, non contact lens wearers and pooled and protease inhibitors were added. 50 u,l of pooled tears were applied to an isoelectric focusing strip containing an immobilised pH gradient (IPG) from pH 3 to 10. When the separation was complete, the strip was attached to a 20 x 23 cm preparative SDS Polyacrylamide 8 to 18% gradient gel for electrophoresis. The gels were electroblotted onto PVOF membranes and stained with amido black. Spots were excised, and subjected to Edman sequencing for the first 4 residues, using a Beckman-Tag Sequenator. The spots were also acid hydrolysed and the amino acid composition was determined. This information, combined with the isoelectric point (pi) and molecular weight of the protein, allowed searching and identification from the Swiss-Prot database. Results: The resolution of the gel was high and at least 10 groups of isoforms were observed. So far, two isoform groups have been analysed. The first represents four isoforms (18-19 kDa, pis 4.2-6.0), and has been identified as lipocalin. Two of these proteins had undergone N-terminal processing. The second is a group of three isoforms of 28 to 29 kDa, with identical sequence of the first 4 residues and pis from 4.4 to 4.8, for which no match could be found. Conclusions: Preparative 2D PAGE is a powerful technique for the separation and identification of tear proteins. So far, two isoform groups have been analysed, and a match for lipocalins obtained in the database. Further analyses will enable the generation of a 2D map of tear proteins from normals, which can be compared with tear protein maps from other physiological and pathological states.