METHOD OF DETECTING O-GLYCOSYLATED PROTEINS IN CELL HOMOGENATES PREPARED FOR PROTEOMIC AND PHOSPHOPROTEOMIC ANALYSIS Russian patent published in 2014 - IPC C12Q1/00 G01N33/50 

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology and a method of detecting O-glycosylated proteins in cell homogenates that are prepared for proteomic and phosphoproteomic analysis. The disclosed invention can be used to perform proteomic and phosphoproteomic analysis. The method involves performing two-dimensional electrophoresis, followed by identification of spots using MALDI-TOF spectroscopy and phosphoproteomic techniques. The cell homogenates are desalinated by gel-penetrating chromatography or dialysis. The cell homogenates are subjected to glycosylation based on a β-elimination principle in a 0.05 M NaOH solution which contains 38 mg/ml NaBH₄ for 16 hours at +45°C, followed by addition of cyanine dye JC-1 in concentration of 10^-6 M. The cell homogenates are incubated for 15 minutes at room temperature. The homogenates are concentrated by precipitation with 50% acetone, subjected to two-dimensional electrophoresis to form electrophoregrams which are analysed for fluorescence when illuminated on a blue light transilluminator with an amber light filter, which visually appears in form of strips which are fluorescent in the dark. Said strips are extracted from the gel and used to perform proteomic or phosphoproteomic analysis. Further analysis of intensity and arrangement of the extracted strips is performed by comparing silver nitrate-coloured electrophoregrams of homogenates before and after a deglycosylation procedure.

EFFECT: disclosed invention enables to identify proteins which change their composition or degree of O-glycosylation as a result of any physiological action on the cell.

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