| Summary: | 碩士 === 國立臺灣師範大學 === 化學系 === 99 === Cerebrospinal fluid (CSF) is an important specimen to accurately reflect pathological processes and provides an ideal window for insights into mechanisms and detection of biochemical changes, such as protein biomarker associated with neurodegenerative disorders and spinal cord injury. However, the wide dynamic range, low protein concentration, and presence of high abundant albumin in CSF pose challenges for comprehensive proteome identification. To facilitate the identification of disease biomarker candidates, we integrated nanoprobe-based albumin depletion technology with iTRAQ or label-free quantitation for quantitative analysis of personalized CSF proteome. The anti-albumin immobilized magnetic nanoparticles (anti-albumin@MNPs) showed 1.4-fold increase in the number of protein identification than commercially available kits to deplete the high abundant albumin in CSF. Combining with iTRAQ or Label-free quantitation, the two platforms provide similar accuracy (mean = 0.003 and -0.066 for iTRAQ and label-free quantitation, respectively in log2 scale) and reproducibility (standard deviation = 0.27 and 0.3 for iTRAQ and label-free quantitation, respectively in log2 scale)
In the second part of thesis, we applied these two strategies to analyze the expression levels of CSF proteome for two diseases: (1) patients with cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) in comparison with age-matched controls and (2) patients with spinal cord injury (SCI) after first and second FGF treatments. A total of 311 proteins were quantified and 39 proteins showed differential expression in more than 60% of CADASIL patients. Among them, amyloid precursor protein, Apolipoprotein E, angiotensinogen, Alpha-1-acid glycoprotein 1, and Alpha-1-acid glycoprotein 2 have been reported to be related to NOTCH3, the disease-causive mutant gene in CADASIL. Further validation using western blot analysis confirmed the down-regulation of amyloid precursor protein in CSF samples from CADASIL patients.
For the CSF analysis for patients with SCI, 233 proteins were identified and 221 proteins were quantified in 7 paired SCI samples from patients after first and second FGF treatments. The preliminary analysis revealed that the FGF treatment induced differential expression of 19%-45% of quantified proteins for the 7 paired SCI patients. Among these proteins, 31 proteins showed common differential expression in more than 4 of SCI patients before and after operation. Four proteins, including Apolipoproteins, tresferrin, tubulin, Zinc finger proteins, had been reported to relate to spinal cord injury.
Our method demonstrated the potential on the identification of potential biomarker for CADASIL disease and the treatment efficiency of SCI.
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