Abstract
Purpose: :
The dynamic distribution of proteins in tears has a vital role in the maintenance of the ocular surface. However, little is known about the PRPs of tear fluid although it is vastly distributed and has been proven as biomarker, which makes these proteins interesting candidates for in-depth proteomics analysis.
Methods: :
Tear samples of 20 volunteers were collected by microcapillary tube and analyzed individually. Proteomic analysis was employed for total protein profiling and characterization with particular interest on PRPs based on 1st & 2nd Dimensional Electrophoresis (DE) and Reversed-phase (RP)-RP 2D capillary LC MALDI TOF/TOF Mass Spectrometry (MS) with data acquisition from WarpLC software (Bruker Daltonics).
Results: :
A total of 70 unique proteins can be distinctly identified and an average of 10% of total proteins is PRPs based on the semi quantitative MS approach. Further characterization of tear protein in 2nd DE showed a total of 12 spots related to PRPs with three distinguished molecular weights of 19, 23 and 26 kDa, and pI values in the range between 5.5 to 3. Existence of wide polymorphism of PRPs reflect their post-translational modifications (PTMs), especially phosphorylation. Unique peptides from the PRPs rich region and all 12 spots identified from the 1st and 2nd DE, showed four distinguished types of peptide combinations from 1601, 1729, 1792 & 1920 m/z for all samples analyzed individually. Sequencing results from tandem MS showed that all four peptides are differentiated by the positions of Glutamine (Q) amino acid. Combinations of these peptides yield Proline Rich Protein 4 (PROL4_HUMAN), pHL E1F1 (gi 1050983) and proline rich 4 (lacrimal) isoform 2 (gi 154448886) proteins from NCBI and SwissProt databases by MASCOT (Matrix Science) search.
Conclusions: :
This work represents one of the most extensive proteome profiles of PRPs generated hitherto from tear fluids by taking advantage of the 2nd DE and MS approaches to identify the PRPs PTMs and classify the PRPs into four different peptide combinations. The recurrence of these different types of PRPs in individuals and its potential as biomarkers for specific pathology is yet to be determined.
Keywords: proteomics • cornea: tears/tear film/dry eye • protein modifications-post translational