優化高含量蛋白去除策略用於全面性分析人類血液蛋白體

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优化高含量蛋白去除策略用於全面性分析人类血液蛋白体

An optimized high abundant protein depletion strategy for large-scale profiling of human blood proteome

陈宜虹 , 硕士　　导师：韩嘉莉　　共同指导教授：张伟峤

繁体中文

血清；血浆；蛋白移除；蛋白质体； Serum ； Plasma ； Protein depletion ； Proteomics

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摘要 │ 参考文献 (26) │ 国际计量

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Among various clinical specimens, plasma and serum is an easily accessible sample for discovery of biomarker candidate proteins associated with disease progression or treatment response. However, the wide dynamic range and complex compositions in plasma/serum present great challenges to identify low abundant proteins of clinical importance. To overcome these bottlenecks, we proposed an optimized sample preparation strategy, which emphasized on the depletion of high abundant proteins in plasma and serum. Specifically, three kinds of antibody-based depletion kits including Aurum serum protein mini kit, ProteoPrepR 20 plasma immunodepletion kit, and Multiple affinity removal spin cartridge human-14, and three antibody-free precipitation methods (ACN, TCA/acetone, and PEG) were evaluated in this study. Plasma samples from kawasaki disease patients and pooled serum samples from 9 fibromyalgia patients were used as references for comparison. 
For single depletion by antibody-based kits, the MARS kit achieved higher (~95%) removal of high abundant proteins compared to ProteoPrep20 (80%) and Aurum (61.4%) and facilitated more identified peptides and proteins. For single depletion by antibody-free methods, TCA/acetone precipitation could remove 68% of protein amount and increase the 1.16-fold number of identified peptides and 1.14-fold proteins compared to raw serum. A significant sample loss was observed by using ACN precipitation with lower identified proteins (0.68-fold) compared to raw serum. Longer preparation time was required for sequential precipitations by multiple PEG concentration which may lead to higher quantitation variation. Based on the single depletion results, we selected MARS and TCA/acetone precipitation as tandem depletion to sequentially deplete high abundant proteins in serum. The results showed that tandem depletion which integrated first MARS depletion and second TCA/acetone precipitation would significantly decrease (from 71.2% to 0.6%) the abundances of top 14 high abundant proteins and increase the number of identified peptides (2.18-fold) and proteins (1.24-fold) compared to raw serum. 
The optimized tandem depletion strategy was applied to quantitative analysis of proteome profiles in serum samples from fibromyalgia patients and healthy controls. Three proteins, FN1, APOA4 and C1QA, were identified with elevated expressions and C3 and APOH were lower expressed in fibromyalgia patients. Among these proteins, FN1, which is an extracellular matrix structural constituent and have been reported to involve in glomerulopathy with fibronectin deposits 2 disease, showed 12.12-fold higher expression in fibromyalgia patients. Through the completion of this study, we expect that our optimized tandem depletion would be an efficient workflow for large-scale quantitative profiling of blood proteome.

参考文献 ( 26 ) 〈TOP〉

1. Anderson, N.L., The Human Plasma Proteome: History, Character, and Diagnostic Prospects. Molecular & Cellular Proteomics, 2002. 1(11): p. 845-867.
连结：
2. Jon M. Jacobs, Joshua N. Adkins, Wei-Jun Qian, Tao Liu, Yufeng Shen, David G. Camp II, and Richard D. Smith, Utilizing Human Blood Plasma for Proteomic Biomarker Discovery. Journal of Proteome Research 2005, 4, 1073-1085
连结：
3. Barh, D., et al., A novel in silico reverse-transcriptomics-based identification and blood-based validation of a panel of sub-type specific biomarkers in lung cancer. BMC Genomics, 2013. 14 Suppl 6: p. S5.
连结：
4. Lista, S., et al., Blood and plasma-based proteomic biomarker research in Alzheimer's disease. Prog Neurobiol, 2013. 101-102: p. 1-17.
连结：
5. Baker, E.S., et al., Mass spectrometry for translational proteomics: progress and clinical implications. Genome Med, 2012. 4(8): p. 63.
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使用DOI作为永久连结

引用含有DOI的文献

DOI可强化引用精确性、增强学术圈连结，并给予使用者跨平台的良好使用经验，目前在全世界已有超过五千万个对象申请DOI。 如想对DOI的使用与概念有进一步了解，请参考 （ ） 。

DOI可强化引用精确性、增强学术圈连结，并给予使用者跨平台的良好使用经验，目前在全世界已有超过五千万个对象申请DOI。如想对DOI的使用与概念有进一步了解，请参考（）。