Detections Of Protein Phosphorylation And Alkaline Phosphatase Activity Based On Nanopore Single-molecule

Protein phosphorylation is one of the most common and important post-translational modifications of proteins in living organisms.Identification of phosphorylation is essential to elucdate complex cellular processes and disease mechanisms.The process of protein phosphorylation and dephosphorylation is dynamically reversible,and the ratio of phosphorylated protein to unphosphorylated protein is usually very low in vivo.In the previous detection process,phosphorylated protein was often covered by the background signal of unphosphorylated protein,so phosphorylated protein was often unable to be effectively analyzed.Alkaline phosphatase?ALP?plays an important role in the process of protein phosphorylation and dephosphorylation.In addition,its activity level can affect a variety of tissues and organs,and it is an indicator of early disease screening in clinical diagnosis.Nanopore single molecule technology has the advantages of high sensitivity,high selectivity,lable-free,and can be used to analyze and detect individual molecules at the level of single molecule.In this paper,we have used amino-modified?-cyclodextrin as a molecular adapter for biological nanopore?-hemolysin??HL?,and have realized the detection of phosphorylated and unphosphorylated proteins and real-time monitoring of ALP activity levels.The main research contents of this paper are as follows:1. Using biological nanopore to detect protein phosphorylationWe have developed a method to detect protein phosphorylation at single molecule level.We used?HL-?M113R?₇-am₇CD as the biological nanopore sensor and oligopeptide containing eight amino acids as the model peptide to detect the phosphorylation of tyrosine at single site.The results showed that there were significant differences in blocking amplitude and dwell time between phosphorylated peptide and unphosphorylated peptide.In addition,the dwell time of tyrosine phosphopeptides at different sites was significantly different.Interestingly,we found that alkaline phosphatase hydrolyzed the C-terminal and N-terminal phosphopeptides at a initial rate 20 times that of intermediate tyrosine phosphopeptides.Our results further deepen the understanding of the mechanism of protein phosphorylation and have certain signficance for the exploration of biological genomics and diseases.2. Real-time monitoring of alkaline phosphatase activity at the single-molecule levelALP plays an important role in dephosphorylation and phosphorylation related cellular regulation and signal transduction.Therefore,it is of great significance to develop an efficient method for the determination of ALP activity in clinical diagnosis.We established a method for measuring ALP activity at the single molecule level based on determining the frequency of disodium 4-nitrophenylphosphate?PNPP?.This method has the advantages of simplicity,rapidity,label-free,high sensitivity and high signal-to-noise ratio.ALP activity was measured in the range of 0-260 U/L.It was found that there was a good linear relationship between ALP activity and PNPP event frequency in the range of 40-200 U/L,with the detection limit down to 0.1 U/L.In addition,we found that macromolecular crowding agents inhibited ALP hydrolysis and magnesium ions promoted ALP hydrolysis.The method established in this paper is used to further realize the screening of ALP inhibitors,which is of great significance for clinical diagnosis and drug screening...