以質譜法分析(1)乙二醛誘導形成的DNA交聯產物 (2)人類血紅蛋白因乙二醛、甲基乙二醛、過氧化亞硝酸所形成的轉譯後修飾，以及 (3)人類尿蛋白的硝化與亞硝化之轉譯後修飾

Mass spectrometric analysis of (1) glyoxal-induced DNA cross-links, (2) glyoxal-, methylglyoxal-, peroxynitrite-induced post-translational modifications of human hemoglobin, and (3) nitrated and nitrosylated human urinary proteins.

陳育進(Yu-Chin Chen)

指導教授 ： 陳皓君

國立中正大學/理學院/化學暨生物化學系/博士(2012年)

摘要

乙二醛和甲基乙二醛是誘導突變物質，會與生物分子反應。乙二醛會與DNA反應，造成DNA的交聯產物，如dG-gx-dC、dG-gx-dG和dG-gx-dA。在此研究中，我們利用毛細管液相層析奈電噴灑串聯式質譜儀，分析在人類胎盤DNA中dG-gx-dC、dG-gx-dG和dG-gx-dA的含量。除此之外，比較兩種不同的水解DNA方法，明顯地看到這三種交聯產物含量的不同。
乙二醛和甲基乙二醛也會與蛋白質反應，造成蛋白質的轉譯後修飾。在此研究中，我們分別以乙二醛、甲基乙二醛與人類血紅蛋白反應，然後以高解析度質譜儀準確的偵測分子量與LTQ的MS2、MS3鑑定轉譯後修飾位置。在人類血液樣品中，鑑定出12個轉譯後修飾位置，包含8個乙二醛和4個甲基乙二醛的修飾，並且相對定量其修飾的程度。
在發炎的過程中，會產生大量的一氧化氮，進而形成過氧化亞硝酸根，造成蛋白質的硝化。在本研究中，我們以過氧化亞硝酸根與人類血紅蛋白反應，然後同樣地以高解析度質譜儀準確的偵測分子量與LTQ的MS2、MS3鑑定轉譯後修飾位置。共鑑定出三個酪胺酸被硝化、一個酪胺酸被亞硝化、三個甲硫酸胺形成亞楓、三個半胱胺酸形成磺酸和一個半胱氨酸形成亞磺酸。相對定量20個吸菸者和20個非吸菸者的血液中血紅蛋白的轉譯後修飾程度的結果顯示α-Tyr-24和α-Tyr-42的硝化程度與抽菸有正相關。，可以探討分析人類血紅蛋白的這些轉譯後修飾做為偵測體內的硝化壓力的生物指標的可能性。
過氧化亞硝酸不僅會造成酪胺酸的硝化，也會造成色胺酸與半胱胺酸的硝化。在本研究中，利用兩種抗3-硝基酪胺酸的抗體，經過免疫沉澱法純化人類尿蛋白中的硝化蛋白，一維電泳的分離，最後奈升流速液相層析串聯質譜分析。目前為止，在人類尿蛋白中，鑑定出25個含硝化或亞硝化蛋白。

關鍵字

奈升流速液相層析串聯式質譜法

並列摘要

Glyoxal and methylglyoxal are mutagens and they react with biological molecules. Glyoxal reacts with DNA to generate DNA crosslinked products, include dG-gx-dC, dG-gx-dG, and dG-gx-dA. In this study, we use capillary liquid chromatography nanospray ionization tandem mass spectrometry (capLC-NSI/MS/MS) assay to quantify the levels of dG-gx-dC, dG-gx-dG, and dG-gx-dA in human placental DNA. We compared two enzyme hydrolysis conditions, and significantly different adduct levels were observed.
Glyoxal and methylglyoxal also react with proteins, resulting in the posttranslational modifications of proteins. In this study, we use glyoxal and methylglyoxal to treat hemoglobin, and the sites of modifications were characterized by accurate mass measurement and by their MS2 and MS3 spectra obtained by LTQ. In human blood samples, we have identified 12 post-translational modifications, included 8 glyoxal- and 4 methylglyoxal-induced modifications and quantified the levels for this post-translational modifications relative to the reference peptides.
Nitric oxide is over-produced during inflammatory processes, and then the formation of peroxynitrite, resulting in the nitration of proteins. In this study, we use peroxynitrite to treat hemoglobin, and the sites of modifications were characterized by accurate mass measurement and by their MS2 and MS3 spectra obtained by LTQ. We have identified nitration on three tyrosine residues, nitrosylation on one tyrosine, sulfoxide formation on all three methionine residues, sulfonic acid on all three cyseine and sulfinic acid on one cysteine. The post-translational modifications were semi-quantified in 20 smokers’ and nonsmokers’ blood samples. The results showed a statistically significant positive correlation between cigarette smoking and the relative quantification of tyrosine nitration at α-Tyr-24 and α-Tyr-42. Thus, measurement of thes PTMs in hemoglobin might be used as feasible biomarker for assessment of nitrative stress in vivo.
Peroxynitrite not only reacts with tyrosine but also with tryptophan and cysteine. The nitrated proteins of human urinary proteins were enriched by immunoprecipitation with two types of anti-nitrotyrosine antibodies, fractionated by 1D SDS PAGE, and analyzed by nanoLC/MS/MS. So far, we have identified 25 nitrated and nitrosylated proteins in human urinary proteins.

並列關鍵字

nanoLC/MS/MS

參考文獻

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