Institute of Bioanalytical Chemistry, Faculty of Chemistry and Mineralogy, Universität Leipzig, Deutscher Platz 5, 04103 Leipzig, Germany.
Analyst. 2013 Sep 7;138(17):5081-8. doi: 10.1039/c3an00724c. Epub 2013 Jul 5.
Mass spectrometry (MS) of 'carbonylated proteins' often involves derivatization of reactive carbonyl groups to facilitate their enrichment, identification and quantification. Among the many reported reagents, 2,4-dinitrophenylhydrazine (DNPH), biotin hydrazide (BHZ) and O-(biotinylcarbazoylmethyl) hydroxylamine (ARP) are the most frequently used. Despite their common use in carbonylation research, their reactivity towards protein-bound carbonyls has not been quantitatively evaluated in detail, to the best of our knowledge. Thus we studied the reactivity and specificity of these reagents towards different classes of reactive carbonyl groups (e.g. aldehydes, ketones and lactams), each being represented by a synthetic peptide carrying an accordingly modified residue. All three tagging reagents were selective for aliphatic aldehydes and ketones. Lactams and carbonyl-containing tryptophan oxidation products, however, were labelled only at low levels or not at all. Whereas DNPH derivatization was efficient under the published standard conditions, the derivatization conditions for BHZ and ARP had to be altered. Acidic conditions provided quantitative labelling yields for ARP. Peptides derivatized with DNPH, BHZ and ARP fragmented efficiently in tandem mass spectrometry, when the experimental conditions were chosen carefully for each reagent. Importantly, the tested carbonylated peptides did not cross-react with amino groups in other proteins present during sample preparations or enzymatic digestion. Thus, it appears favourable to digest proteins first and then derivatise the reactive carbonyl groups more efficiently at the peptide level under acidic conditions. The carbonylated model peptides used in this study might be valid internal standards for carbonylation proteomics.
蛋白质羰基的质谱分析（MS）通常涉及对反应性羰基的衍生化，以促进其富集、鉴定和定量。在众多报道的试剂中，2,4-二硝基苯肼（DNPH）、生物素酰肼（BHZ）和 O-（生物素羰酰基甲基）羟胺（ARP）是最常用的试剂。据我们所知，尽管它们在羰基研究中被广泛使用，但它们对蛋白质结合羰基的反应性尚未被详细定量评估。因此，我们研究了这些试剂对不同类型反应性羰基基团（如醛、酮和内酰胺）的反应性和特异性，每个基团都由携带相应修饰残基的合成肽来代表。所有三种标记试剂都对脂肪族醛和酮具有选择性。然而，内酰胺和含有羰基的色氨酸氧化产物仅在低水平或根本不被标记。虽然 DNPH 衍生化在已发表的标准条件下是有效的，但 BHZ 和 ARP 的衍生化条件必须改变。酸性条件为 ARP 提供了定量标记产率。当为每个试剂仔细选择实验条件时，用 DNPH、BHZ 和 ARP 衍生化的肽在串联质谱中有效地碎裂。重要的是，测试的羰基化肽不会与样品制备或酶消化过程中存在的其他蛋白质中的氨基发生交叉反应。因此，似乎有利的是先消化蛋白质，然后在酸性条件下在肽水平上更有效地衍生化反应性羰基基团。本研究中使用的羰基化模型肽可能是羰基蛋白质组学的有效内标。
