以質譜法分析(一)在糖尿病患者與大腸癌患者的血紅蛋白上的氯化、硝化與氧化修飾; (二)在大腸癌患者的血紅蛋白上的穀胱甘肽修飾

Mass Spectrometry Analysis of (1) Chlorination, Nitration, and Oxidation In Hemoglobin of DM and Colon Cancer Patients And (2) Glutathionylation In hemoglobin of Colon Cancer Patients.

楊雅雰(Ya-Fen Yang)

指導教授 ： 陳皓君

國立中正大學/理學院/化學暨生物化學系/碩士(2015年)

摘要

體內過量具反應性物種的產生會傷害生物體，並與糖尿病、大腸癌等疾病有相關性。本研究使用奈升流速液相層析奈電噴灑游離質譜法鑑定出氯化劑和溴化劑修飾人類血紅蛋白的位置，並同時測量氯化、溴化、硝化、氧化等修飾的程度。我們分析了19個第二型糖尿病患者和18個正常人，結果顯示糖尿病患者的血紅蛋白在α-Tyr-24修飾上氯、α-Tyr-42修飾上硝基和 α-Met-7修飾上氧的平均含量皆高於正常人。我們也分析了14個大腸癌患者和15個正常人血紅蛋白上這些修飾，結果顯示大腸癌患者的血紅蛋白在α-Tyr-24修飾上氯、α-Tyr-42修飾上硝基和 α-Met-76修飾上氧的平均含量皆高於正常人，並具統計學上之意義。因此，希望藉由量測血紅蛋白上這些轉譯後修飾可以作為糖尿病和大腸癌低侵入性的生物指標候選人。基於血液樣品採集與儲存的便利性，我們評估血紅蛋白上這些修飾的穩定性，我們從採血紙上將血紅蛋白萃取出來，並分析血紅蛋白上氯化、硝化及氧化修飾的程度。發現在14天內兩種存放條件(室溫和4 °C)下的所有修飾的含量之相對誤差介於0.44%~16.4%。

榖胱甘肽 (glutathione, GSH)會和細胞內蛋白質上的半胱胺酸(cysteine) 側鏈反應產生雙硫鍵的鍵結，這種反應稱為榖胱甘肽化反應 (glutathionylation)，是一個重要的蛋白質轉譯後修飾。而這種蛋白質的修飾也已被證明會影響蛋白質的結構和功能，且與癌症具相關性。本研究使用奈升流速液相層析奈電噴灑游離串聯質譜法分析在血紅蛋白上3個半胱胺酸位置經穀胱甘肽化的程度。結果顯示大腸癌患者的血紅蛋白在α-Cys-104和β-Cys-93修飾上穀胱甘肽平均含量皆高於正常人，並具統計學上意義。因此，希望藉由量測血紅蛋白上這些轉譯後修飾可以作為糖尿病和大腸癌低侵入性的生物指標候選人。

關鍵字

大腸癌 ； 糖尿病 ； 血紅蛋白

並列摘要

Endogenous reactive species have the potential to damage tissues, and they are implicated in diseases including diabetes and colorectal cancer. This study identified the sites of chlorination and bromination in human hemoglobin and measured the extent of chlorination, bromination, nitration ,and oxidation by nanoflow liquid chromatography–nanospray ionization tandem mass spectrometry (nanoLC-NSI/MS/MS) under the selected reaction monitoring (SRM) mode. Semiquantification of these modifications in hemoglobin extracted from blood samples shows that the extents of chlorination at α-Tyr-24, nitration at α-Tyr-42, and oxidation at α-Met-32 and α-Met-76 are significantly higher in poorly controlled type 2 diabetic patients (n=19) than in normal individuals (n=18). In addition, chlorination at α-Tyr-24, nitration at α-Tyr-42, and oxidation at α-Met-32 and α-Met-76 are significantly higher in colorectal cancer patients (n=14) than in normal individuals (n=15). Thus, measuring these PTMs in hemoglobin should provide potential low-invasive biomarker candidates for type 2 diabetes mellitus and colorectal cancer . To evaluate the convenience of blood sample collection and stablility of these modifications in hemoglobin, I extracted hemoglobin from dried blood spot (DBS). The extents of modifications, including chlorination, nitration and nitrosylation of tyrosine as well as oxidation of cysteine and methionine residues, in human hemoglobin were measured in the trypsin digest by nanoLC-NSI/MS/MS under the SRM mode. The extents of all PTMs are stable (RSD 0.44%－16.4%) within 14 days when stored at room temperature and 4℃.
The side chain of cysteine residues in hemoglobin react with glutathione and form disulfide bonds, termed glutathionylation, is an important post-translational modification. Protein glutathionylation has been proven to affect protein structure, cause protein dysfunction, and it is relatded to cancer formation. In this study, we used nanoLC-NSI/MS/MS to measure the extent of glutathionylation at 3 cysteine sites in human hemoglobin. Using this method, we found that the extents of glutathionylation at α-Cys-104 and β-Cys-93 in hemoglobin from 16 colorectal cancer patients were significantly higher than those in 13 normal Individuals with a p value of 0.0455 and 0.0351, respectively

並列關鍵字

Diabetes mellitus ； Colorectal cancer ； Hemoglobin

參考文獻

1. Fu, X.; Wang, Y.; Kao, J.; Irwin, A.; d'Avignon, A.; Mecham, R. P.; Parks, W. C.; Heinecke, J. W., Specific sequence motifs direct the oxygenation and chlorination of tryptophan by myeloperoxidase. Biochemistry. 2006, 45 (12), 3961-71.

2. Zhang, S. Q.; Tsiaras, W. G.; Araki, T.; Wen, G.; Minichiello, L.; Klein, R.; Neel, B. G., Receptor-specific regulation of phosphatidylinositol 3'-kinase activation by the protein tyrosine phosphatase Shp2. Mol. Cell. Biol. 2002, 22 (12), 4062-72.

3. Leeuwenburgh, C.; Rasmussen, J. E.; Hsu, F. F.; Mueller, D. M.; Pennathur, S.; Heinecke, J. W., Mass spectrometric quantification of markers for protein oxidation by tyrosyl radical, copper, and hydroxyl radical in low density lipoprotein isolated from human atherosclerotic plaques. J. Biol. Chem. 1997, 272 (6), 3520-6.

4. Knutson, C. G.; Mangerich, A.; Zeng, Y.; Raczynski, A. R.; Liberman, R. G.; Kang, P.; Ye, W.; Prestwich, E. G.; Lu, K.; Wishnok, J. S.; Korzenik, J. R.; Wogan, G. N.; Fox, J. G.; Dedon, P. C.; Tannenbaum, S. R., Chemical and cytokine features of innate immunity characterize serum and tissue profiles in inflammatory bowel disease. Proc. Natl. Acad. Sci. U. S. A. 2013, 110 (26), E2332-41.

5. Hazen, S. L.; Heinecke, J. W., 3-chlorotyrosine, a specific marker of myeloperoxidase-catalyzed oxidation, is markedly elevated in low density lipoprotein isolated from human atherosclerotic intima. J. Clin. Invest. 1997, 99 (9), 2075-81.