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Krishnatej Nishtala, Lei Zhou, Rohit Shetty, Kiran kumar, Roger W. Beuerman, Arkasubhra Ghosh; Screening of tear molecular markers for early detection of keratoconus by quantitative mass spectrometry. Invest. Ophthalmol. Vis. Sci. 2017;58(8):3531.
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© ARVO (1962-2015); The Authors (2016-present)
To screen and identify changes in tear fluid proteome that can act as diagnostic markers for early detection in patients with progressing grades of keratoconus(KC) by label-free targeted quantitative mass spectrometry
Tear proteins extracted off Schirmers strips collected from patients at grades I-III (n= 33,25 and 21) of KC and healthy control subjects(n=40) were subjected to proteolytic digestion prior to differential labelling by iTRAQ. For screening the disease associated changes, only proteins quantifiable in atleast half the patients across the clinical grades of KC are considered. Disease specific alterations in the tear proteome was screened by building secondary ratios of proteins between the lower and higher grades of comparison. A ratio fold difference of ≥1.5 and a two-tailed t-test p value≤0.05 between the grades of comparison is considered to be statistically significant. The differential proteins displaying either an elevated or reducing trend in progressing grades of KC were validated in an independent cohort of patients, control(n=28) and grades I-III (n=23, 25 and 13) by parallel reaction monitoring(PRM). Each patient tear fluid sample was spiked with 5fmol/µl of E.coli beta-galactosidase digest as an internal standard
iTRAQ analysis identified altered expression of around 200 proteins in increasing grades of KC. Panther pathway analysis showed glycolytic proteins, proteins on Rho GTPase cytoskeletal regulation, p38/MAPK pathway and FGF signaling to be significantly altered in KC patients 35 proteins satisfying the filter criteria of ratio fold ≥1.5 and two-tailed t-test p value≤0.05 are considered as potential markers for progression of keratoconus. Proteins lacritin (LACRT), cystatin-s(CST4), lactoperoxidase(LPO), Ig alpha 1 chain(IGHA1) were identified to be elevated whereas tear proteins lysozyme C(LYSC), lipocalin1(LCN1) were among those with reduced levels in increasing clinical grades of KC. So far nine proteins-LACRT, LYSC, LCN1, DMBT1, SG2A1, ANXA1, 1433Z, CYTN, and IGHA1 were validated with atleast 1 peptide/protein by PRM in an independent cohort of 21(total 90) patients(con-3, GI-III 6 each)
Using quantitative mass spectrometry, our study identified a panel of molecular markers that help in screening the progression of KC which can aid in early diagnosis and in distinguishing the disease from other ocular surface conditions
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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