| Alzheimer's disease(AD)is an age-related neurological disease affecting millions of people around the world.Its main pathological hallmarks are insoluble deposits of ?-amyloid(A?)and hyperphosphorylated tau proteins,which form extracellular plaques and intracellular neurofibrillary tangles,respectively.The detailed mechanism of this process is still unknown.However,one definitive result of protein aggregation is synaptic dysfunction and neuronal loss in Alzheimer's disease brain.Mitochondrial dysfunction,oxidative stress,inflammation,and metal metabolism are all thought to be involved in this process.Metal ions such as copper,zinc and iron all play an important role in brain function in both health and disease states.Changes in the content and distribution of metal ions in the body are common features of different neurodegenerative diseases and aging.Alzheimer's disease signature proteins and their metal-binding properties may be the basis of important biochemical processes in pathophysiological processes.In this project,we established a method for the absolute quantification of A? protein in brain tissue.Quantification of low levels of AP1-40 is a significant indicator in Alzheimer's disease research.The interaction between the three antibodies M3.2,4G8 and 11A50B of A?1-40 protein was used for immunoprecipitation.The acid(pH=2)elution was performed at 70? and combined with MALDI-TOF MS.13C labeled A?1-40 protein was as an internal standard.The recovery rate of A?1-40 standard protein was up to 48.7%.There was no significant difference(p>0.1)in the content of AP1-40 in the homogenate of mouse brain tissue measured by this method and by ELISA.This method has the advantages of simple operation,good repeatability and high accuracy.It is more suitable for proteins which do not have corresponding ELISA kits on the market.An ID-ICP-MS method was also established to determine the contents of iron,copper,and zinc in the cortex,hippocampus,thalamus,and other brain regions of mouse brain.The detection limit,limit of quantitation,precision and repeatability were also determined.The results showed that compared with the control mice,the contents of iron,copper and zinc in brains of model rats were significantly different in different brain regions.Copper and zinc were mainly deposited in the hippocampus and cortex of mice,while iron was mainly deposited in the mouse hippocampus.We also determined the protein content in brain regions of the model group and the control group by ELISA assay to explore the relationship between Alzheimer's disease-related proteins and metals.The results showed that A?1-40 protein,A?1-42 protein,APP protein and tau protein were all found in the hippocampus and cortex,and the content in the model group was higher than that in the control group.The distribution is similar to the distribution of iron,copper,and zinc in various brain regions.However,the content of metallothionein-3(MT-3)in each brain area of the model group was lower than that of the control group,and there was no significant difference in the contents of each brain area.By consulting the literature,the resulting relationship between the various proteins and metals was explained in terms of mechanism.In conclusion,we have established two new methods to quantify the protein in mouse brain tissue and metal elements in mouse brain regions.We also explored the content relationship between protein and metal in mouse brain regions.This thesis provided a new insight for exploring the relationship between metal and protein in Alzheimer's disease and quantitative study of disease markers. |
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