Biomarker Detection And Analysis Based On Mass Spectrometry

Mass spectrometry has outstanding performance in sensitivity and suitability for identification of structure determination of trace substances in complex systems characteristics, and also has advantages in accuracy, operability, detection speed, and universality on the analysis of proteins and other biological activity molecules. However, mass spectrometry LC-MS-based studies are time-consuming and MALDI-TOF MS-based research, although with short time-consuming, the preparation process is complex and the sample transfer is low. What’s more, there is no research-based MALDI Shangqie quantitative Detection of-TOF MS in mass rapid detection and we can only do a limited sample of qualitative research. In order to solve the above problem, the following research was carried out:(1) A kind of method based on characteristics of peptides with meat animal traceability has been explored. Combined research-based proteomics on MALDI-TOF, LC-MS and other advanced detection equipment with a common market Confusing-meat (chicken, pigs, cattle, sheep, duck) as experimental subjects, after a certain amount of pre-treatment, meat extracts myoglobin. Through the extracted myoglobin be drawn difference MALDI-TOF MS analysis of five myoglobin. The use of peptide sequence tag technology compares different animal meat hydrolyzed polypeptide sequence information, and is established to detect meat traceability analysis method based on the characteristics of peptides. Experimental use of liquid chromatography-mass spectrometry techniques to identify the feasibility of different meat confirmed sources, the results show there are five characteristics peptide Confusing-meat hydrolyzed, and these peptides can be used for meat traceability analysis, which established based on MALDI TOF MS and HPLC to build a method of meat traceability detection and identification.(2) A quantitative detection method based on physical MALDI-TOF MS biomarkers is established, regarding brain natriuretic peptide (BNP) and angiotensin II (Angiotensin II) as biomarkers and adding some internal standards which has similar physical and chemical properties. In the experimental testing process of type B natriuretic peptide (BNP), it added some internal standards which has similar chemical and physical properties to lysine B-type natriuretic peptide (BNP+K). The results showed that the signal strengths of BNP and BNP+K, which have different concentration ratios, have a good linear relationship. Based on this, the method for quantitative detection of the target sample has been built up, especially for biomarker detection. For the experiment, angiotensin II results showed that based on the MALDI-TOF MS method for quantitative determination of the verification result indicates that the method is feasible. And can be extended to use for other quantitative detection of biomarker s.(3) A micro-column method for preparing a protein-based surface modification and coupling has been established. And a method for preparing of capillary affinity column used for MALDI-TOF MS was investigated. The hydroxyl group was introduced on the inner-face using "Piranha" solution. Amino group was coupled on the capillary by the reaction between3-aminopropyl-triethoxysilane (APTES) and hydroxyl group. Konjac glucomannan (KGM), activated with1,4-butanediol diglycidyl ether (BDDE), was conjugated on the capillary by the reaction between the epoxy group on the KGM and-NH2on the capillary. Then model protein (trypsin) was conjugated on the activated KGM. The effects of KGM molecular weight and the ratio of epoxy group to the KGM on the trypsin conjugation were determined. The results indicated that the KGM molecular weight was the key factor affecting the quantity of conjugated trypsin. With the trypsin inhibitor as target protein, the capillary affinity column could be used for desalting or sample concentrating. The strategy for preparing capillary affinity column, to be used for sample pretreatment prior to MALDI-TOF MS analysis, was confirmed.The paper developed the method of an array of micro-chromatography mass spectrometry for improving the efficiency to ensure the process of transfer with high flux, high stability and high repeatability. This article is created based on MALDI-TOF MS DE quantitative detection method and the sample traceability LC-MS-based method, which pointed out a new direction for the application of the bio-mass spectrometry technology.