Identification of carbonylated peptides by tandem mass spectrometry using a precursor ion-like scan in negative ion mode
Identification of carbonylated peptides by tandem mass spectrometry using a precursor ion-like scan in negative ion mode
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DOI：
10.1016/j.jprot.2011.05.033
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发表时间：
2011-10-19
影响因子：
3.3
通讯作者：
Hoffmann, Ralf
中科院分区：
文献类型：
--
作者：
Bollineni, Ravi Ch;Fedorova, Maria;Hoffmann, Ralf
活性氧（ROS）几乎可以氧化蛋白质的任何氨基酸残基。尽管某些修饰在细胞内是可逆的，但较高的氧化态尤其是不可逆的。这些不可逆的翻译后修饰被广泛用作氧化应激的生物标志物，例如蛋白质羰基化，将醛、酮和内酰胺称为“反应性羰基”。本研究依赖于一组含有 C 末端醛（精氨酸）或在赖氨酸或组氨酸残基处用丙酮酸（酮）或 4-羟基壬烯醛（醛）修饰的合成肽，以及含有焦谷氨酸（脯氨酸的氧化产物）和 2-氨基-3-丁酸（氧化产物）的肽 苏氨酸）。羰基化位点专门用 2,4-二硝基苯肼 (DNPH) 衍生化，并在电喷雾电离 (ESI-) MS 中研究了产物的裂解行为。重要的是，DNPH 标记的羰基化肽在负离子模式 ESI 中表现出良好的电离行为，提供了一种灵敏的检测方法。在这些条件下，常规肽大多被区分。在针对带负电的离子测试的裂解技术中，脉冲 Q 解离提供了 m/z 值 152.0、163.1 和 179.0 的三种诊断离子，对 DNPH 修饰的肽具有特异性。这些标记离子成功地应用于检测牛血清白蛋白和从 HeLa 细胞获得的复杂蛋白质混合物的加标胰蛋白酶消化物中的羰基化模型肽。 (C) 2011 Elsevier B.V. 保留所有权利。
Reactive oxygen species (ROS) can oxidize proteins at almost any amino acid residue. Whereas some modifications are reversible within the cells, the higher oxidation states are especially irreversible. These irreversible post translational modifications are widely used as biomarkers of oxidative stress, such as protein carbonylation, which refers to aldehydes, ketones and lactams as 'reactive carbonyl groups'. This study relied on a set of synthetic peptides containing a C-terminal aldehyde (arginal) or modification with pyruvic acid (ketone) or 4-hydroxynonenal (aldehyde) at lysine or histidine residues, as well as peptides containing pyroglutamic acid (oxidation product of proline) and 2-amino-3-butyric acid (oxidation product of threonine). The carbonylation sites were specifically derivatized with 2,4-dinitrophenylhydrazine (DNPH) and the fragmentation behavior of the products investigated in electrospray ionization (ESI-) MS. Importantly, the DNPH-labeled carbonylated peptides showed favorable ionization behaviors in negative ion mode ESI providing a sensitive detection method. Regular peptides were mostly discriminated under these conditions. Among the fragmentation techniques tested for the negatively charged ions, pulsed Q dissociation provided three diagnostic ions at m/z values 152.0, 163.1 and 179.0, specific for DNPH-modified peptides. These marker ions were successfully applied to detect the carbonylated model peptides in a spiked tryptic digest of bovine serum albumin and a complex protein mixture obtained from HeLa cells. (C) 2011 Elsevier B.V. All rights reserved.