Characterization of a new qQq-FTICR mass spectrometer for post-translational modification analysis and top-down tandem mass Spectrometry of whole proteins
Characterization of a new qQq-FTICR mass spectrometer for post-translational modification analysis and top-down tandem mass Spectrometry of whole proteins
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DOI：
10.1016/j.jasms.2005.08.008
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发表时间：
2005-12-01
影响因子：
3.2
通讯作者：
O'Connor, PB
中科院分区：
文献类型：
--
作者：
Jebanathirajah, JA;Pittman, JL;O'Connor, PB
本文介绍了一种新的电喷雾qQq傅里叶变换离子回旋质谱仪（qQq-FTICR MS）在生物学方面的应用。该qQQ-FTICR质谱仪设计用于研究后修饰蛋白质和自上而下分析生物相关蛋白质样品。磷酸化，一个常见的和重要的翻译后修饰的分析仪器的效用进行了研究。选择磷酸化作为一个例子，因为它是普遍存在的，具有挑战性的分析。此外，探索了使用该仪器进行蛋白质的自上而下测序，因为该仪器为该方法提供了特别的优势。对不同的蛋白质进行自上而下的测序，包括市售蛋白质和生物来源的样品，如人E2泛素缀合酶UbCH 10。获得了人UbCH 10的良好序列标签，允许明确鉴定蛋白质。该仪器由商业生产的前端构建：聚焦仅射频四极杆（Q 0），随后是分辨四极杆（Q1）和LINAC四极杆碰撞室（Q2），与FTICR质量分析仪组合。它在分析亚化学计量浓度的样品中具有实用性，因为离子可以在ICR池中分析之前在质量分辨Q1中分离并在Q2中积累。在LCMS兼容的时间尺度上证明了Q2冷却和片段化的速度和功效，并且证明了磷酸肽在10阿莫尔/μ L范围（pM）内的检测限。该仪器的目的是使几个破碎方法，包括撇渣破碎，Q2碰撞活化解离（Q2 CAD），多极存储辅助解离（MSAD），电子捕获解离（ECD），红外多光子诱导解离（IRMPD），和持续的非共振辐射（SORI）CAD，从而允许各种MSn实验。该系统的一个特别有用的方面是使用Q1从复杂的混合物中分离离子，其具有小于Im/z的窄隔离窗口。这些功能使自上而下的蛋白质分析实验以及复杂混合物的次要组分的结构表征成为可能。
The use of a new electrospray qQq Fourier transform ion cyclotron mass spectrometer (qQq-FTICR MS) instrument for biologic applications is described. This qQq-FTICR mass spectrometer was designed for the study of post-translationally modified proteins and for top-down analysis of biologically relevant protein samples. The utility of the instrument for the analysis of phosphorylation, a common and important post-translational modification, was investigated. Phosphorylation was chosen as an example because it is ubiquitous and challenging to analyze. In addition, the use of the instrument for top-down sequencing of proteins was explored since this instrument offers particular advantages to this approach. Top-down sequencing was performed on different proteins, including commercially available proteins and biologically derived samples such as the human E2 ubiquitin conjugating enzyme, UbCH10. A good sequence tag was obtained for the human UbCH10, allowing the unambiguous identification of the protein. The instrument was built with a commercially produced front end: a focusing rf-only quadrupole (Q0), followed by a resolving quadrupole (Q1), and a LINAC quadrupole collision cell (Q2), in combination with an FTICR mass analyzer. It has utility in the analysis of samples found in substoichiometric concentrations, as ions can be isolated in the mass resolving Q1 and accumulated in Q2 before analysis in the ICR cell. The speed and efficacy of the Q2 cooling and fragmentation was demonstrated on an LCMS-compatible time scale, and detection limits for phosphopeptides in the 10 amol/mu L range (pM) were demonstrated. The instrument was designed to make several fragmentation methods available, including nozzle-skimmer fragmentation, Q2 collisionally activated dissociation (Q2 CAD), multipole storage assisted dissociation (MSAD), electron capture dissociation (ECD), infrared multiphoton induced dissociation (IRMPD), and sustained off resonance irradiation (SORI) CAD, thus allowing a variety of MSn experiments. A particularly useful aspect of the system was the use of Q1 to isolate ions from complex mixtures with narrow windows of isolation less than I m/z. These features enable top-down protein analysis experiments as well structural characterization of minor components of complex mixtures.