Comparative Analysis Of Nano-SiO₂Induced Alteration On Membrane Proteins In HaCaT Cells

Objective:To investigate the effects of nano-silica (nm-SiO2) exposure on the expression of membrane proteins in HaCaT cell and screen improtant biomarkers to reveal the nm-SiO2-induced toxicological mechanisms.Methods:A ProteoExtract Subcellular Proteome kit was used to extract membrane proteins of HaCaT cells. A method combined with nano-liquid chromatography and matrix-assisted laser desorption time of flight mass spectrometry (nano-LC-MALDI-TOF-MS)were used for membrane proteins sepeartion and identification. A comparative mem b-rane proteomics was preformed by two-dimensional fluorescence difference gel electrop hor-esis (2D-DIGE) and MALDI-TOF-MS analysis. A bioinformatics analysis was used to identify the subcellular localization and molecular function of identified proteins.Results:(1) A total of15proteins were identified by nano-LC-MALDI-TOF-MS, and9of them are considered with membrane-associated in swissprot database. The ratio is60%, demonstrating that the methodology is useful.(2) the morphological analysis and cell activtiy assay suggested that the concentration of10mg/L15-nm SiO2for24h exposure is the best condition for proteomic study.(3) A comparative membrane proteomic study were performed by two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) and matrix-assisted laser desorption time of flight mass spectrometry (MALDI-TOF-MS) in the membrane fraction of HaCaT cells. A total of17proteins were identified in this analysis and6of them are considered as membrane-associated in the swissprot database.Conclusions:(1) the development of an effective and feasible method of extraction of membrane proteins and membrane proteomics research.(2) A comparative membrane proteomics was developed in combination with two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) and matrix-assisted laser desorption time of flight mass spectrometry (MALDI-TOF-MS) to analyze the membrane fraction in HaCaT cell under nm-SiO2exposure.