iTRAQ Quantitative Proteomics in the Analysis of Tears in Dry Eye Patients

Abstract

PURPOSE. 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.METHODS. 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 ¼ 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 lg 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). RESULTS.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).CONCLUSIONS. 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. (Invest Ophthalmol Vis Sci. 2012; 53:5052-5059) DOI:10.1167/iovs.11-9022 T he Dry Eye Workshop in 2007 defined dry eye (DE) as a multifactorial ocular surface disease diagnosed by symptoms of discomfort, and signs of visual disturbance, tear film instability, and ocular surface damage, accompanied by increased osmolarity of the tear film and ocular surface inflammation.1 It is evident from the definition that the tear film and ocular surface are altered in DE disease. The tear film serves/performs a variety of functions and is composed of various substances, including proteins, lipids, mucins, salts, and other organic molecules.2 The aqueous component constitutes the majority of the tear layer, and the proteins in the tear film are believed to have a key role in the protection of the external surface from potential pathogens, and also are involved in modulation of wound healing process. [3][4][5][6] The major source of tear proteins is the secretory acinar cells of the lacrimal gland, including the primary proteins of the tear film: lyso...

Omics Biomarkers in Ophthalmology

“…Interestingly, on protein, metabolite, and lipid levels, specific expressions for subgroups of dry eyes based on disease severity have been identified. [128][129][130] These markers might therefore function as indicators of disease progression, although further validation is required to confirm these results.…”

Tissue Transglutaminase Is a Negative Regulator of Monomeric Lacritin Bioactivity

“…Twenty-three to 25 kDa lacritin is 50% less, 7-fold less, or at an average ratio of 0.24 of normal, respectively, in blepharitis, 27 contact lens-related dry eye, 26 and aqueous deficient dry eye. 28 One study reported no lacritin change, but the methodology substituted 2-D SDS-PAGE for liquid chromatographic cationic separation followed by reverse phase separation. 29 While characterizing a new generation of monoclonal antilacritin N-(1F5-C9-F4; '1F5') and rabbit C-('ab C-term') domain-specific polyclonal antibodies, we unexpectedly discovered~75 kDa lacritin immunoreactive material in human tear and saliva samples that previously had not been subjected to antilacritin immunoblotting.…”

Diabetic retinopathy: Proteomic approaches to help the differential diagnosis and to understand the underlying molecular mechanisms

“…Chemical labeling with iTRAQ or TMT makes possible the labeling of up to 8 or 10 different samples with an isobaric label [38][39][40][41][42][43]. The label is constructed in that way that the m/z values of the peptides originating from different samples (precursor ions) are the same but during fragmentation the labeling group is released and the rest of the peptide can be used for MS/ MS based protein identification.…”