High Accuracy Quantitative Study Of One Of The Biomarkers Of Alzheimer’s Disease----tau Protein

Alzheimer’s disease（AD）is a neurodegenerative disease.At present,many biomarkers have been discovered,among which tau protein is one of the most important biomarkers of AD.Abnormal tau protein phosphorylation is an important features of AD,since there is no effective drugs,so the earlier detected,the lower critically ill patients.Early detection requires more accurate detection methods to avoid missed diagnosis or misdiagnosis,it is very important to establish a kind of high accuracy test method of tau protein.In this study,purified and recombinant full-length Tau protein（441）was taken as the research object.Before the test,tau protein was divided into 100μg/m L single samples and stored in the freezer at-80℃.A reliable method for accurate quantification of Tau protein was explored through the same batch of samples.Explore the influence of different objects on detection results and detection methods,and verify the consistency of different methods.The experimental methods and results are as follows:The stable characteristic peptide obtained by enzymatic hydrolysis was used as the detection target to achieve the determination of Tau protein.After the optimization of trypsin addition amount and hydrolysis time,the ratio of enzyme addition amount to protein is 1:10,and the enzymolysis time should be more than 48 h.Before the enzymatic hydrolysis,the labeled characteristic peptides were introduced to participate in the enzymatic hydrolysis,and the peak area ratio of the labeled peptides to the common peptides was detected.The accurate concentration of Tau protein solution can be obtained by calculation,and the stability of the method and peptide segment can be tested by multiple experiments.Finally,the value of Tau protein in simple matrix was achieved,and the value was 75.08±3.00μg/m L.Tau protein was fully hydrolyzed into amino acids by the method of amino acid hydrolysis,and the value analysis of Tau protein was realized through the detection and value determination of target amino acids.In the experiment of amino acid hydrolysis,the temperature,concentration of hydrochloric acid,the amount of hydrochloric acid added and the hydrolysis time were optimized respectively.Finally,130℃was selected and 1.2m L of 6mol/L hydrochloric acid was added to obtain the results after treatment and the time of reaction is48 hours.Hydrolysis before joining theory quality than approximately 1:10marker amino acid,the experiment selected the five kinds of amino acid,valine,proline,alanine,leucine and phenylalanine,hydrolysis in LC-MS spectrometer corresponding amino acid was detected in the normal and standard of the peak area ratio of amino acids,the formulas to accurate concentration of tau protein can be obtained.After several experiments,the value of Tau protein was 74.47±3.00μg/m L.High performance liquid chromatography（HPLC）and high resolution inductively coupled plasma mass spectrometer（HR-ICP-MS）were used to combine the separation advantages of chromatography with the detection advantages of mass spectrometry to achieve the detection and determination of Tau protein through the detection and determination of sulfur element in protein.In the experiment,shimadzu high performance liquid chromatograph auxiliary pump was used to pump 0.1 ppm ³⁴S isotope thinner stably into the three-way mixing tube at a flow rate of 0.15m L/min.After mixing with the sample pipeline,it entered the high-resolution ICP-MS.By detecting the signal response intensity of ³²S and ³⁴S,the protein mass spectrum peak was obtained.The accurate concentration of Tau protein in the sample to be tested can be obtained after calculation according to the mass flow formula.After several experiments,the value of Tau protein is 74.13±3.00μg/m L.The value of the three experimental methods is relatively consistent,and the results are still stable after many tests,and the stability of the method and the sample is also proved.An experimental method for high accuracy quantitative analysis of Tau protein in simple matrix was established.