Dynamic Mutations And Tumor Marker Related Analysis Method Study Based On Biological Mass Spectrometry

Biological mass spectrometry have been wide used in the fields of health,medicine,biology,environment,food,clinical and others.Studies have shown that when applied in qualitative and quantitative analysis of protein and nucleic acid,mass spectrometry can improve the accuracy of the detection results significantly,and reduce the detection limit and the error rate as well.Therefore,it is urgent to develop some simple and accurate methods for rapid diagnosis,treatment and pro-diagnosis monitoring diseases and tumors by mass spectrometry/mass spectrometry or chromatography-mass spectrometry.In this paper,a variety of molecular biology techniques combined with mass spectrometric detection to reach the goal.We take Huntington’s disease,carcinoembryonic antigen,prostate-specific antigens as our detected objects.The mainly contents of this paper are described as follows:1.Based on PCR amplification techniques and high specificity of magnetic capture,we propose a concentration of nucleic acid analysis method by PCR amplification,beads capture,acid hydrolysis,mass spectrometry analysis[In Chapter 2].In details,two primers were designed for PCR amplification,magnetic beads with streptavidin was used for enrichment of the target chain marked with biotin,after that,double-stranded DNA were denatured into single-strand after treat with high temperature 5 minutes,acid hydrolysis of single strand into single bases,Take the natural existence base thymine whose number is constant as the internal standard,cytosine whose mass signal is highest among the bases as the detect object,we analysis it quantitatively in MS/MS mode.The DNA chain with different repeat sequence take the C/T signal as the vertical coordinate,CAG repeat number as the horizontal to establish the standard curve,we finally achieve a sensitive detection of real samples.The detective sensitivity is up to femto molar level.2.Based on the characteristics of liposome,for example,simply prepared,stable,and with good biological compatibility.We developed a method using liposome encapsulated gold nanoparticles,the lipid was modified with PEG-N3 cross-linking with carcinoembryonic antigen(CEA)and prostate-specific antigen(PSA)antibody modified with DBCO-PEG4-NHS by click chemical,At the same time,the protein G beads were incubation with antibody,the target protein was captured by using specific antibody-antigen recognition,then the sandwich structure of antibody-antigen-antibody were formed,and finally the sample would qualitative and quantitative by MADLI-TOF-MS[In chapter 3].The high ionization efficiency of phospholipids made them easy to be detected,the quantity of two proteins CEA,PSA can be detected indirectly by detecting the phospholipid which was combined with the antibody of the two proteins.The specificity of the method was high,and the results were not affected by other proteins.Add phospholipids as the internal standard with constant concentration for the quantitative of different concentrations of antigen analysis,and then established the standard curve.Limit of detection of CEA was 0.37 ng/mL and PSA was 0.06 ng/mL,and the results of serum samples were approximately the same as the analysis of that of the ELISA kit.