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Ka Wai, Jimmy CHEUNG, Jingfang Bian, Fengjuan Yu, King Kit Li, Chi Ho To, Lei Zhou, Thomas Chuen Lam; Comprehensive proteomic study of chick vitreous during normal growth. Invest. Ophthalmol. Vis. Sci. 2018;59(9):2357. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Despite being the largest component of the eye, vitreous humor (VH) is often overlooked at in research as it is mostly consisted of water. With recent breakthrough in LCMS-based profiling techniques, protein identification and quantification in the VH has emerged as an important area for studying various ocular diseases such as myopia. Using chick as an animal model, this study aims to establish a vitreous proteome library as well as quantifying differential expressed proteins during the normal growth using label free mass spectrometry approach with Sequential Windowed Acquisition of All Theoretical Fragment Ion Mass Spectra (SWATH).
Normal growing (7,14,21,28 days old) White Leghorn Chicks were employed for this study. A 12/12-hour dark/light cycle was used to represent the normal light cycle. After refraction and ocular parameters measurements, chicks were sacrificed and eyeballs were removed and hemisected immediately to harvest the VH. VH samples were homogenized with T-PER lysis buffer (Thermo) using a homogenizer (Bertin). Equal amount of proteins in samples were prepared and digested with trypsin. Pool samples for RE and LE respectively were analyzed by Nano LC-MS/MS with a TripleTOF 6600 mass spectrometer (SCIEX). Data obtained were exported and analyzed using ProteinPilot 5.0, PeakView 2.2 and MarkerView 1.3 (SCIEX) . Functional classification was analyzed using PANTHER classification system.
Refraction and vitreous chamber depth (VCD) were found to be significantly changed (P<0.01,n=18) during the normal growth period at each time point. A total of 1260 non-redundant protein (17605 distinct peptides) were identified from the pool sample consisting all time points of chicks VH at 1% FDR. With the stringent filtering criteria set as cut off threshold at >2 folds, differential expression in the same direction from both eyes and each identified protein must have at least 2 quantifiable peptides: 3 proteins were found up regulated and 9 proteins were found down regulated across all time points (day 14,21,28) compared to the baseline at day 7.
Here we demonstrated a proteomic workflow for chick vitreous during normal growth (At Day 7, 14, 21 and 28). A large amount of proteins was identified in this comprehensive protein library during this normal growth period. Most differential expressed proteins quantified from this study were found to be novel structural/ growth-related proteins.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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