Single eye analysis and contralateral eye comparison of tear proteins in normal and dry eye model rabbits by MALDI-ToF mass spectrometry using wax-coated target plates

Abstract

A study of rabbit tear protein expression in a dry eye rabbit model was performed to determine if a pattern in expressed proteins could be identified. The uniqueness of the model allows the comparison of normal (control) eye tear protein expression with surgically induced dry eye tear protein expression in individual animals. The sensitivity of the method allows for single eye analysis. One-dimensional mini-gel electrophoresis of the tear proteins did not show substantial differences between band patterns of the normal versus the dry eye, but was used to assess the molecular weight ranges of the major proteins. Specific assignments of some of the predominant proteins were obtained by tandem mass spectrometry (MS) which showed that the lower molecular weight lipid-binding proteins (approximately 10 kDa to 36 kDa) constitute a considerable amount of the observed protein, followed in lesser quantities by the transferrins which have higher molecular weights ranging from 70 kDa to 85 kDa. Enhancement of matrix-assisted laser desorption/ionization time-of-flight (MALDI-ToF) MS linear mode analysis of intact proteins in tear fluid was demonstrated through the use of wax-coated MALDI plates and spot washing. MALDI-ToF MS analysis of the expressed tear proteins illustrates that differences between normal eye tear and dry eye tear protein content are manifested in changes in the lower molecular weight lipid-binding proteins such as lipophilin which exhibits an increase in concentration in the dry eye, and beta-2 microglobulin which undergoes a decrease.

Comparison of two methods of tear sampling for protein quantification by Bradford method

“…The Bradford method is suggested as a sensitive method of tear protein quantification (Sitaramamma et al 1998, Ham et al 2007, Ananthi et al 2008), as we observed. Previous comparisons between the Lowry and Bradford methods for the quantification of tear proteins showed significant differences between both and higher values attributed by the Bradford method, alerting to the need of a careful interpretation of the results (Lu et al 2010).…”

“…In our study the samples were frozen at -80 o C (Zhou et al 2003, and stored for no longer than 2 months. It was proved that samples stored at -70°C (Sitaramamma et al 1998, Ham et al 2007, Yoon et al 2010) for up to 4 months preserved the tear proteins better than in 4°C or -20 o C in a shorter time, when it was observed a reduction in the proteins of the sample (Sitaramamma et al 1998).…”

“…Petznick et al (2011) analyzed eye-flush tears method in cats and suggested to collect pooling eye-flush tears for tear analysis that need high total protein content. Nevertheless, the method of collection can be challenging, especially in individuals with dry eye (Ham et al 2007), which encourages the use of STT strip by some researchers (Li et al 2008).…”

Lacrimal Proline Rich 4 (LPRR4) Protein in the Tear Fluid Is a Potential Biomarker of Dry Eye Syndrome

“…Selective defect in aquaporin 5 (AQP5) trafficking is seen in patients with sjogren’s syndrome [30]. Lipophilin was shown to be significantly increased in the dry eye relative to the normal as studied in rabbit model [31]. Most of these proteins are high abundant proteins of the tear.…”

A Novel MALDI Matrix for Analyzing Peptides and Proteins: Paraffin Wax Immobilized Matrix

“…[24][25][26] Compared to the hydrophobic layer above, paraffin wax coating was easy to prepare. Furthermore, it also offered some other advantages 25 including (1) low cost and easy sample preparation, (2) more homogeneous coverage of matrix/analytes over the sample surface, (3) inert surface that minimized side reactions, and (4) easy removal after analysis.…”