Trelle Morten B, Jensen Ole N
Department of Biochemistry and Molecular Biology and Centre for Epigenetics, University of Southern Denmark, DK-5230 Odense M, Denmark.
Anal Chem. 2008 May 1;80(9):3422-30. doi: 10.1021/ac800005n. Epub 2008 Mar 14.
Tandem mass spectrometry (MS/MS) is a powerful tool for characterization of post-translationally modified proteins, including epsilon-N-acetyllysine-containing species. Previous reports indicate that epsilon-N-acetyllysine immonium ions are useful marker ions for peptides containing epsilon-N-acetyllysine, but the specificity and sensitivity of these ions for assignment of lysine acetylation by MS/MS have not been studied in detail. We investigated MS/MS data sets of 172 epsilon-N-acetyllysine tryptic peptides and 268 nonacetylated tryptic peptides to establish the utility and reliability of epsilon-N-acetyllysine immonium ions for identification and validation of acetylated peptides. Our analysis shows that the immonium ion at m/z 143 lacks specificity for lysine-acetylated peptides, whereas the derivative at m/z 126 is highly specific (98.1%). We also studied the positional effect of the epsilon-N-acetyllysine on the intensity of observed acetyllysine immonium ions. We observed an increase in acetyllysine immonium ion intensities when the acetylated lysine was N-terminally positioned in the peptide as compared to internal positions. Based on these observations we propose a validation scheme for unambiguous assignment of acetyllysine-containing peptides by MS/MS. Our analysis of epsilon-N-acetyllysine immonium ions provide a framework for investigation of MS/MS marker ion specificity and sensitivity that can be applied in studies of other types of post-translational modifications.
串联质谱（MS/MS）是用于表征翻译后修饰蛋白质的强大工具，包括含ε-N-乙酰赖氨酸的物种。先前的报道表明，ε-N-乙酰赖氨酸亚铵离子是含ε-N-乙酰赖氨酸肽段的有用标记离子，但这些离子通过MS/MS用于赖氨酸乙酰化鉴定的特异性和灵敏度尚未得到详细研究。我们研究了172个含ε-N-乙酰赖氨酸的胰蛋白酶肽段和268个非乙酰化胰蛋白酶肽段的MS/MS数据集，以确定ε-N-乙酰赖氨酸亚铵离子用于鉴定和验证乙酰化肽段的实用性和可靠性。我们的分析表明，m/z 143处的亚铵离子对赖氨酸乙酰化肽段缺乏特异性，而m/z 126处的衍生物具有高度特异性（98.1%）。我们还研究了ε-N-乙酰赖氨酸位置对观察到的乙酰赖氨酸亚铵离子强度的影响。我们观察到，与内部位置相比，当乙酰化赖氨酸位于肽段的N端时，乙酰赖氨酸亚铵离子强度会增加。基于这些观察结果，我们提出了一种通过MS/MS明确鉴定含乙酰赖氨酸肽段的验证方案。我们对ε-N-乙酰赖氨酸亚铵离子的分析为研究MS/MS标记离子的特异性和灵敏度提供了一个框架，可应用于其他类型翻译后修饰的研究。
