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I.-J. Wang, Y.-H. Chen, T.-H. Young; Proteomic Approach to Study the Difference of Protein Expression Between Adhesive and Spheroid Keratocytes. Invest. Ophthalmol. Vis. Sci. 2008;49(13):4299.
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© ARVO (1962-2015); The Authors (2016-present)
The object of this study was to compare the difference of protein expression between these cultured keratocytes and keratocyte spheroids by proteomic approaches.
Primary bovine keratoytes were incubated with DMEM/10%HS on chitosan-coated surface or tissue culture plate at 37 °C for 7 days. Cell lysates were prepared by lysis buffer and total proteins were extracted. Isoelectric focusing (IEF) was carried out on an Ettan IPGphorisoelectric focusing system by using 13-cm IPG strips (pI 3-10). Protein (75µg) was added to rehydration solution. Strips were rehydrated for 6 h and then IEF was performed for a total of 24,500 Vh. SDS-PAGE (12.5%) was applied for the second-dimension electrophoresis. The analysis of image was using ImageMaster 2D Elite 3.10 software (Amersham Bioscience, Piscataway, NJ) In-gel digestion and mass spectrometry, protein spots were destained with 25-mM ammonium bicarbonate and dehydrated with 50% acetonitrile. After the peptides were eluted with trifluoracetic acid of various concentrations, the peptide mixtures were subjected to mass spectrometric analysis. A QStar (Applied Biosystems, Foster City, CA) hybrid quadrupole time-of-flight (QqTOF) mass spectrometer equipped with a matrix-assisted laser desorption ionization (MALDI) source and a nitrogen laser (337 nm) was used to acquire MALDI-MS and -MS/MS spectra. The MS/MS spectra were used to automatically search the NCBI protein database using MOSCOT software.
In 1-D SDS-PAGE with silver stain, high level expression of the ~25kDa protein and the ~70kDa protein for keratocyte spheroids/aggregates were found with lower cell activity in them. In contrast, higher level expression of ~100kDa protein was observed in keratoyctes incubated on tissue culture plate. The pI of the 25kDa, the 70kDa , and the 100kDa protein were about 5.5, 6.8, and 6.3, observed by 2-D electrophoresis. Analysis of mass spectrometry show that the 25kDa and 70kDa were manganese superoxide dismutase (MnSOD) and heat shock 70kDa protein (Hsp 70) respectively. However, the 100kDa protein did not fit any protein which has been established in the database.
The high level expression of MnSOD and Hsp70 might refer that the cell in spheroids form is an environment with higher stress.
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