iTRAQ quantitative proteomics in the analysis of tears in dry eye patients.

Sruthi Srinivasan, Mirunalni Thangavelu, Liwen Zhang, Kari B Green, Kelly K Nichols

Investigative ophthalmology & visual science 2012 Jul 31

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We analyzed the change in protein expression of tear film proteins in dry eye (DE) and non-DE (NDE) patients using isobaric tag for relative and absolute quantitation (iTRAQ) technology. We categorized 24 participants into NDE, and mild (MDE), moderate-to-severe (MSDE), and mixed (MXDE) DE on the basis of clinical DE tests. Tear samples (n = 6 subjects/group) were collected using Schirmer's strips. Proteins were extracted from strips and were quantified using the Bradford assay. Protein from each sample was pooled as internal standard (IS), and 20 μg protein from each sample and the IS were digested and labeled with different tandem mass tag (TMT) isobaric mass tag labeling reagent. The reaction was quenched and the labeled peptides were mixed. Samples were injected for liquid chromatography-mass spectrometry (LC/MS/MS) analysis on the Orbitrap mass spectrometer. Bioinformatic analyses were performed using protein information resource (PIR). Combined results showed a total of 386 proteins in tears as determined by the iTRAQ experiments. An average of 163 proteins was detected in each of 6 biologic replicates. Of those, 55% were detected 6 times and 90% were detected multiple times (>2). In addition to the down-regulation of commonly reported proteins, such as lipocalin-1, lysozyme, and prolactin-inducible protein across all sub groups of DE, a number of proteins were significantly differentially regulated in MSDE and other subgroups of DE. A greater number of proteins were down-regulated in MSDE versus MDE, and the specific functions involved include response to stimulus (8 vs. 6 proteins), immune system process (6 vs. 4), regulation of biologic processes (3 vs. 3), and ion transport (2 vs. 2). 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 DE patients. PIR is a useful resource to interpret pathways and functions of proteins.

Citation

Sruthi Srinivasan, Mirunalni Thangavelu, Liwen Zhang, Kari B Green, Kelly K Nichols. iTRAQ quantitative proteomics in the analysis of tears in dry eye patients. Investigative ophthalmology & visual science. 2012 Jul 31;53(8):5052-9