Rauniyar Navin, Stevens Stanley M, Prokai-Tatrai Katalin, Prokai Laszlo
Department of Molecular Biology & Immunology, University of North Texas Health Science Center, 3500 Camp Bowie Boulevard, Fort Worth, Texas 76107, USA.
Anal Chem. 2009 Jan 15;81(2):782-9. doi: 10.1021/ac802015m.
Reactive oxygen species generated during oxidative stress can lead to unfavorable cellular consequences, predominantly due to formation of 4-hydroxy-2-nonenal (HNE) during lipid peroxidation. Data-dependent and neutral loss (NL)-driven MS(3) acquisition have been reported for the identification of HNE adducts by mass spectrometry-based proteomics. However, the limitation associated with this method is the ambiguity in correct assignment of the HNE modification site when more than one candidate site is present as MS(3) is triggered on the neutral loss ion. We introduce NL-triggered electron capture dissociation tandem mass spectrometry (NL-ECD-MS/MS) for the characterization of HNE-modification sites in peptides. With this method performed using a hybrid linear ion trap-Fourier transform ion cyclotron resonance (FTICR) mass spectrometer, ECD in the FTICR unit of the instrument is initiated on precursor ions of peptides showing the neutral loss of 156 Da corresponding to an HNE molecule in the prescan acquired via collision-induced dissociation tandem mass spectrometry in the linear ion trap. In addition to manifold advantages associated with the ECD method of backbone fragmentation, including extensive sequence fragments, ECD tends to retain the HNE group during MS/MS of the precursor ion, facilitating the correct localization of the modification site. The results also suggest that predisposition of a peptide molecular ion to lose HNE during collision-induced dissociation-based fragmentation is independent of its charge state (2+ or 3+). In addition, we have demonstrated that coupling of solid-phase enrichment of HNE-modified peptides facilitates the detection of this posttranslational modification by NL-driven strategies for low-abundance proteins that are susceptible to substoichiometric carbonylation during oxidative stress.
氧化应激过程中产生的活性氧可导致不良的细胞后果，这主要是由于脂质过氧化过程中形成了4-羟基-2-壬烯醛（HNE）。基于质谱的蛋白质组学已报道了数据依赖和中性丢失（NL）驱动的MS(3)采集用于鉴定HNE加合物。然而，该方法的局限性在于，当存在多个候选位点时，由于在中性丢失离子上触发MS(3)，HNE修饰位点的正确归属存在模糊性。我们引入了NL触发的电子捕获解离串联质谱（NL-ECD-MS/MS）来表征肽中的HNE修饰位点。使用混合线性离子阱-傅里叶变换离子回旋共振（FTICR）质谱仪进行该方法时，仪器FTICR单元中的ECD在前体离子上启动，这些前体离子在通过线性离子阱中的碰撞诱导解离串联质谱获得的预扫描中显示出对应于一个HNE分子的156 Da中性丢失。除了与ECD主链断裂方法相关的多种优势，包括广泛的序列片段外，ECD在前体离子的MS/MS过程中倾向于保留HNE基团，有助于修饰位点的正确定位。结果还表明，肽分子离子在基于碰撞诱导解离的碎片化过程中失去HNE的倾向与其电荷状态（2+或3+）无关。此外，我们已经证明，HNE修饰肽的固相富集耦合有助于通过NL驱动策略检测这种翻译后修饰，该策略适用于在氧化应激期间易发生亚化学计量羰基化的低丰度蛋白质。
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