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Absolute quantitative analysis of intact and oxidized amino acids by LC-MS without prior derivatization

机译：LC-MS无需先前衍生化的完整和氧化氨基酸的绝对定量分析

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The precise characterization and quantification of oxidative protein damage is a significant challenge due to the low abundance, large variety, and heterogeneity of modifications. Mass spectrometry (MS)-based techniques at the peptide level (proteomics) provide a detailed but limited picture due to incomplete sequence coverage and imperfect enzymatic digestion. This is particularly problematic with oxidatively modified and cross-linked/aggregated proteins. There is a pressing need for methods that can quantify large numbers of modified amino acids, which are often present in low abundance compared to the high background of non-damaged amino acids, in a rapid and reliable fashion. We have developed a protocol using zwitterionic ion-exchange chromatography coupled with LC-MS to simultaneously quantify both parent amino acids and their respective oxidation products. Proteins are hydrolyzed with methanesulfonic acid in the presence of tryptamine and purified by strong cation exchange solid phase extraction. The method was validated for the common amino acids (excluding Gln, Asn, Cys) and the oxidation products 3-chlorotyrosine (3-ClTyr), 3-nitrotyrosine (3-NO2Tyr), di-tyrosine, Nε-(1-carboxymethyl)-l-lysine, o,o’-di-tyrosine, 3,4,-dihydroxyphenylalanine, hydroxy-tryptophan and kynurenine. Linear standard curves were observed over ~3 orders of magnitude dynamic range (2–1000 pmol for parent amino acids, 80 fmol–20 pmol for oxidation products) with limit-of-quantification values as low as 200 fmol (o,o’-di-tyrosine). The validated method was used to quantify Tyr and Trp loss, and formation of 3-NO2Tyr on the isolated protein anastellin treated with peroxynitrous acid, and for 3-ClTyr formation (over a 2 orders of magnitude range) in cell lysates and complex protein mixtures treated with hypochlorous acid.
机译：氧化蛋白损伤的精确表征和定量是由于较低的丰度，繁多和修饰的异质性导致的重大挑战。肽水平（蛋白质组学）的质谱（MS）基础技术提供了由于不完全序列覆盖和不完美的酶消化而产生的详细但有限的图像。这与氧化改性和交联/聚集蛋白特别有问题。对于能够快速可靠的时尚，可以迫切需要量化大量改性氨基酸的方法，这些方法通常与未受损氨基酸的高背景相比常见于低丰度。我们开发了一种使用与LC-MS偶联的两性离子离子交换色谱法的协议，同时定量母体氨基酸及其各自的氧化产物。蛋白质在试色氨酸存在下用甲磺酸水解并通过强阳离子交换固相萃取纯化。该方法验证了常见的氨基酸（不包括Gln，Asn，Cys）和氧化产物3-氯陶器（3-Cltyr），3-硝基曲霉（3-NO2TYR），二酪氨酸，Nε-（1-羧甲基）的方法-L-赖氨酸，O，O'-酪氨酸，3,4，羟基苯基丙氨酸，羟基 - 色氨酸和犬育霉素。观察到线性标准曲线超过〜3个幅度动态范围（2-1000pmol，母氨基酸，80mol-20 pmol用于氧化产物），其限制值低至200 fmol（O，O'-二酪氨酸）。验证的方法用于量化Tyr和TRP损失，并在用过氧酸处理的分离的蛋白anaStellin上形成3-no2tyr，以及在细胞裂解物和复合蛋白质混合物中的3-cltyr形成（在2次幅度范围内）用次氯酸处理。

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