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S. Srinivasan, M. Thangavelu, L. Zhang, K. B. Green-Church, K. K. Nichols; Use of ITRAQ Quantitative Proteomics in the Analysis of Tear Film in Dry Eye Patients. Invest. Ophthalmol. Vis. Sci. 2010;51(13):5200.
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© ARVO (1962-2015); The Authors (2016-present)
To analyze the relative expression of tear film proteins in dry eye (DE) and non dry eye (NDE) patients using ITRAQ Technology (Isobaric Tag for Relative and Absolute Quantitation).
24 participants were categorized into NDE (OSDI score ≤ 12; Schirmer scores (SS) ≥ 16mm), mild DE (MDE- OSDI 13-22; SS: 6 -15mm), moderate to severe DE (MSDE- OSDI ≥24; SS: ≤5mm) and mixed DE (MXDE - OSDI score- variable; SS: 6-15mm). Tear samples (n =6/group) were collected using Schirmer strips. Proteins were extracted from Schirmer strips using 100mM triethylammonium bicarbonate with 400µl 0.05% ProteaseMAX as extraction buffer. Extracted proteins were quantified using Bradford assay. Protein (11µg) from each sample was pooled as internal standard (IS) and 20µg protein from each sample and the IS was digested, labeled with different TMT Isobaric Mass Tag labeling reagent (Thermo Scientific). The reaction was quenched and the labeled peptides were mixed and concentrated to a final concentration of 1 µg/µl. About 1.5 µl of each sample was injected for LC/MSMS analysis on the Orbitrap mass spectrometer (Thermo Scientific). Data search was performed with MASCOT search engine against Swiss-prot human database.
Combined results showed a total of 386 proteins in tears as determined by the ITRAQ experiments. Average of 163 proteins was detected in each of 6 biological replicates. Of those, 55% were detected 6 times and 90% were detected multiple times (>2). In addition to the down-regulation of commonly reported lacrimal gland proteins such as lipocalin-1, lysozyme and prolactin-inducible protein across all sub groups of DE, a list of proteins were significantly differentially regulated in MSDE (Table 1) and other subgroups of DE.
ITRAQ is one of the newest tools for quantitative mass spectrometry in tear proteome research. Differences in the protein ratios can be detected between normal and dry eye patients and continued work to characterize involved pathways is needed. Future work to validate findings using Western blot and ELISA assays are planned.
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