開發整合型醣胜肽分析策略應用於探討血清生物標記蛋白質

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doi:10.6342/NTU201700296

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开发整合型醣胜肽分析策略应用於探讨血清生物标记蛋白质

Development of an Integrated Glycopeptide Analytical Strategy for Serum Protein Biomarker Analysis

陈贞均 , 博士　　导师：翁启惠　　

英文 DOI： 10.6342/NTU201700296

蛋白质醣基化；串联式质谱技术；醣胜肽定量；甲种胎儿蛋白；肝癌； N-linked glycosylation ； intact glycopeptide identification ； mass spectrometry ； targeted quantification ； alpha-fetoprotein ； hepatocellular carcinoma

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Protein glycosylation is one of the most common post-translational modifications and has received increased attention for its critical role in cell biology and diseases.  Despite its biological significance, the delineation of intact glycopeptides and site-specific glycopeptide quantification still await exploration. In this dissertation, we established an analytical workflow integrating a newly-designed automated tool for glycopeptide profiling and an enzyme-assisted targeted glycopeptide quantification to facilitate the understanding of glycosylation in diseases.
First, Mass-spectrometry-based Automated Glyco-peptide IdentifiCation platform (MAGIC), an in-house automated computational tool was developed to accurately characterize the intact glycopeptides without prior knowledge of the proteins and glycans present in the sample. Using a novel “Trident” strategy that detects a triplet pattern corresponding to [Y0, Y1, Y2] or [Y0−NH3, Y0, Y1] from the fragmentation of the common trimannosyl core of N-linked glycopeptides, the Y1 (i.e. peptide+GlcNAc) ion was identified from beam-type collision-induced dissociation tandem mass spectra.  After Y1 assignment, the software generates in silico spectra by overwriting the original precursor with the naked peptide m/z and removing all of the glycan-related ions to identify the peptide sequence by common database search engines. Finally, MAGIC computes the glycan compositions and ranks them. Result from several datasets with various sample complexity from single protein to cell lysate implied that MAGIC enables to identify N-linked intact glycopeptide at a high speed and with high accuracy. 
In the second part, we developed a mass spectrometry based methodology to study the altered core-fucosylation in human sera. In this study, we selected alpha-fetoprotein (AFP) as a model target analyte since its core-fucosylated isoform has been used as a diagnostic and prognostic marker for hepatocellular carcinoma (HCC). To overcome the limitation on quantitative mass spectrometry, we took an advantage of endoglycosidase, which specifically cleaves the glycosidic bond between two GlcNAc residues, to reduce the microheterogeneity of the glycan on peptide backbone. This will result in truncated glycopeptides, e.g. peptide with GlcNAc or peptide with coreFuc GlcNAc, which can be directly analyzed by LC-MS/MS and quantified by extracting the peak area of target m/z. We evaluated the analytical figures of merit of the proposed method by following the guideline of the US Food and Drugs Administration (US FDA) for bioanalytical method validation. The targeted glycopeptide quantitation had good linearity from 15.6-1000 ng/mL (r2=0.9930 for interday experiments) and a limit of detection of 15.6 ng/mL. We anticipate applying the proposed protocol to understand the correlation between glycosylation and liver diseases by calculating the ratio of core-fucosylated to non-core-fucosylated glycopeptides in different patient groups.

参考文献 ( 123 ) 〈TOP〉

[7] a) A. S. Vercoutter‐Edouart, M. C. Slomianny, O. Dekeyzer‐Beseme, J. F. Haeuw and J. C. Michalski, Proteomics 2008, 8, 3236-3256; b) A. Arcinas, T.-Y. Yen, E. Kebebew and B. A. Macher, Journal of proteome research 2009, 8, 3958-3968; c) S. A. Whelan, M. Lu, J. He, W. Yan, R. E. Saxton, K. F. Faull, J. P. Whitelegge and H. R. Chang, Journal of proteome research 2009, 8, 4151-4160.
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[12] Y. Tian and H. Zhang, PROTEOMICS – Clinical Applications 2010, 4, 124-132.
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[14] J. Zhang and D. I. C. Wang, Journal of Chromatography B: Biomedical Sciences and Applications 1998, 712, 73-82.
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[15] B. Buszewski and S. Noga, Analytical and Bioanalytical Chemistry 2012, 402, 231-247.
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[18] A. J. Alpert, Analytical Chemistry 2007, 80, 62-76.
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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（）。