Quantitative Research And Application Of Alzheimer’s Disease Biomarkers Based On Mass Spectrometry

Aβ42(β amyloid peptide,Aβ42)is a β amyloid polypeptide composed of 42 amino acids,which is an important biomarker for Alzheimer’s disease(AD).It was found that the content of Aβ42 in cerebrospinal fluid of the patients decreased significantly.Therefore,the determination of the content of Aβ42 in the cerebrospinal fluid is important for the diagnosis of Alzheimer’s disease.The clinical detection for Alzheimer’s patients is mainly conducted by imaging and immunology.The advantages of these two methods are rapid and convenient.However,neither can be absolutely quantitative,which means that the same patient may have different test results with different methods or different laboratories.Therefore,the establishment of absolute quantitative analysis of Aβ42 and the development of related reference materials are of great significance for the early diagnosis and drug development of AD.The main contents of this work include:(1)The liquid chromatography-isotope dilution mass spectrometry(HPLC-IDMS)was established to achieve accurate quantitative analysis of β amyloid peptide by measuring target amino acids in Aβ peptides.Firstly,by optimizing the hydrolysis time,temperature and acid dosage,the optimal conditions for Aβhydrolysis conditions were determined.Secondly,the ion pair,voltage and collision energy were optimized to determine the mass spectrometry detection conditions of amino acids.Finally,according to the formula of isotope dilution method and the target amino acids content in Aβ,the content of Aβ in amyloid peptide was obtained.(2)The post-column isotope dilution method-inductively coupled plasma mass spectrometry(ID-ICPMS)was established to achieve accurate quantitative analysis of β amyloid peptide by measuring the sulfur element in Aβ.The Aβ42 in pure β amyloid peptide was separated from impurities by optimizing 4 kinds of liquid chromatography conditions.It can be found that the buffer salt system composed of NaH2PO4-Na2HPO4 has a good separation effect on Aβ by separating Aβ42,So the separation of Aβ40 and impurities can be achieved by changing the ratio of the two.In view of the influence of enriched 34S diluent on 32S/34S signal intensity and sensitivity,the flow rate of 34S diluent was optimized.The response factor(f)was introduced to correct the response of mass spectrometry(F)during sample detection.According to the formula of isotope dilution method and the number of S elements in Aβ,the content of Aβin β amyloid peptide was 0.759±0.014 g g-1(Aβ42)and 0.757±0.015 g g-1(Aβ40),respectively.ID-ICPMS research results show that in the range of 0.2-2.2 μg g-1,the linear correlation coefficient is larger than 0.99,the detection limit are 156 pg g-1(Aβ42)and 162 pg g-1(Aβ40),and the limit of quantification are 520 pg g-1(Aβ42)and 540 pg g-1(Aβ40).The recovery rate of the target is between 95%and 106%,and the relative standard deviation is less than 6%.The results of this sample by HPLC-IDMS are 0.761±0.020 g g-1(Aβ42)and 0.760±0.023 g g-1(Aβ40).The measurement results of the two methods are in good agreement.Compared with the HPLC-IDMS analysis method,this method has improved detection limit and sensitivity,and has traceability.(3)The HPLC-IDMS and ID-ICPMS methods were applied to the development of β amyloid peptide reference materials.The established method is used to determine the value of β amyloid peptide standard materials.In order to ensure the stability of the quality value of the standard materials during the user’s use,the homogeneity of the standard material candidates and the stability during storage were investigated.The homogeneity test includes the homogeneity between samples and the homogeneity in the bottle.The F test showed that the homogeneity of the standard substance candidates was good.Stability testing was carried out at certain time intervals.The short-term stability testing temperature was-20 and-4℃,and the long-term stability testing temperature was-70℃.The t test showed that the stability of the standard substance candidate was good.In this work,HPLC-IDMS and ID-ICPMS methods based on mass spectrometry were established to quantify β amyloid peptide,and the optimal hydrolysis conditions of Aβ,mass spectrometry detection conditions of target amino acids,and Aβ liquid chromatography separation conditions were determined.Finally,the quantitative method was applied to the development ofβ amyloid peptide reference material,which can be used for the verification of the Aβ test method,the traceability of the test results and the diagnosis of Alzheimer’s disease.