A Novel Gold Nanoparticles Probe For The Detection Of Glutathione In Living Cells And Verification Of The Phosphoarginine Site Of Proglucagon

Glutathione(GSH)is an important biological antioxidant,involved in a series of cellular biological activities.GSH is considered to be a biomarker of many diseases,including cancer and Alzheimer’s disease.Developing a simple,effective method to detect intracellular GSH is vital for the study of cellular functions and disease diagnosis.In this paper,a bifunctional linker(AEDP)-modified gold nanoparticles probe(Au@PLL-AEDP-FITC)was designed for the detection of GSH in living cells.The fluorescence molecule(FITC)was connected to gold nanoparticles through the synthetic bifunctional linker(AEDP)which containing a disulfide bond,resulting in the FITC fluorescence quenching and the construction of Au@PLL-AEDP-FITC.The disulfide bond was broken by the reaction between GSH and disulfide bond,leading to the recovery of fluorescence signal.The change of fluorescence signal could be used for the determination of GSH.This method shows good linearity with concentrations of GSH from 1 × 10-8 to 1.8 × 10-7 M,providing a detection limit as low as 5.07 × 10-9 M.Au@PLL-AEDP-FITC has high selectivity for GSH and can be used for quantitative detection of GSH in complex biological systems.Owing to a large number of positive charges on the surface of Au@PLL-AEDP-FITC,it can be easily absorbed by cells and successfully applied in GSH fluorescence imaging of HeLa cells.Compared with other GSH detection methods,our method is simple,low cost,high sensitivity and good selectivity.It has great potential in monitoring the change of GSH concentration in living cells and cell imaging.Protein phosphorylation plays a key role in many physiological activities such as cellular signal transduction and protein synthesis.The phosphorylation of arginine,histidine and lysine,is called N-phosphorylation.The research progress of Nphosphorylation is slow due to the P-N bond is highly unstable in acidic media and higher temperatures.Research on phosphoarginine(pArg)has not made a breakthrough until recent few years.134 N-phosphorylated proteins and 217 pArg sites have been identified from Bacillus subtilis.These sites are involved in stress response,protein degradation and cell movement.There are few studies on pArg modification in eukaryotic cells now.In our previous work on the identification of N-phosphorylation proteins and sites in Jurkat cells,we found there is N-phosphorylation modification between Arg71 and His72 on the RHDEFER sequence of proglucagon.Herein,we used potassium phosphoramidate PPA to phosphorylate the peptide(RHDEFER),then the phosphorylate histidine peptide(RpH72DEFER)was separated and purified for Nano LC-MS/MS detection.The MS/MS analysis data of RpH72DEFER peptide did not match the previously mass spectrometry data of the N-phosphorylation peptide.It means that the N-phosphorylation sites on the RHDEFER of proglucagon is Arg71,not His72.Arg71 was important cleavage site of prohormone convertases,the Nphosphorylation of this site may affect the release of glucagon.Therefore,verification of the phosphoarginine site(pArg)is of great significance,providing a basis for further investigation of the influence of pArg modification in sugar metabolism.