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Identification of proteins from tobacco seedlings (Nicotiana tabacum) exposed to silver nanoparticles from 2-D gel electrophoresis using AssayMAP technology

Nanoparticles research is currently an area of intense scientific surveys. Among different NPs, silver NPs are of particular interest because of their antibacterial and antifungal properties. AgNPs can be found in many different consumer products. Since plants play a significant role in accumulation and biodistribution of many environmentally released substances, they could serve as a potential pathway for AgNP-transport and bioaccumulation into food chains. Current proteomics-based techniques have been scarcely used on plants to analyse the effects of AgNPs. Our research goal was to develop the method to identify proteins from 2-D-gels using liquid handling chromatography. Tobacco seedlings were treated with 100μM citrate-AgNP. 10th day of treatment, proteins were extracted and separated on IEF, pH 3-10 NL gradient strips, followed by 2-D-PAGE. Proteins were detected with Coomassie BB. Tryptic peptides were separated and purified using Bravo AssayMAP instrument. 50 protein spots were identified using MALDI- TOF/TOF analyzer. 11 protein spots which were not identified in the first instance were repeated and subjected to 1-D strong cation-exchange fractionation at pH10/pH2.5 and C18 2-D fractionation using AssayMAP instrument. Until now, we successfully fractionated and identified several important but difficult- to-identify proteins especially ones with glycine-rich domains. With this method, we will be able to identify proteins which usually remain unidentified with conventional proteomics approach.

silver nanoparticles; proteomics; 2-D gels; chromatography; tobacco seedlings

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