DETECTION AND ANALYSIS OF PEPTIDES/PROTEINS WITH O-LINKED MODIFICATIONS

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 一些O连接的蛋白修饰是可逆的翻译后修饰，在生物信号通路中至关重要，并在细胞中以亚化学计量浓度存在。这一事实以及质谱仪中在碰撞激活条件下这些修饰的可能性使得它们很难通过传统的串联MS策略进行检测。已经设计了几种检测这些修饰的方法，包括衍生化和标记策略。然而，所有这些方法都存在灵敏度不足的问题。使用β-消除和Michael加成的衍生化方法解决了这个问题，但引入了两个新的问题：i)磷酸化和糖基化竞争相同的位点，并且β-消除的条件很难微调，使得只有一个修饰被衍生，而不是两个都衍生；ii)衍生化方法去除了糖，因此不区分复杂的O-糖基化或单一的O-GlcNAc加成。为了用MS检测这种极不稳定的修饰，分析过程中使用的电离方法和碎裂方法都必须非常“软”，以保持修饰的完整性。本实验室开发的两种仪器(振动冷却MALDI FTMS和ESI-QQQ FTMS)已被证明是O-糖基化和磷酸化分析的理想仪器，因为它们都提供非常软的电离条件，并且离子光学可调，从而使修饰的蛋白质/肽完整地转移到ICR细胞。电子捕获解离(ECD)是诱导多肽断裂的首选方法，因为它不会从多肽侧链上切割修饰。我们已经证实，ECD具有广泛的能力，产生几乎完整的序列信息(脯氨酸除外)，同时不会导致不稳定的侧链修饰的碎片，包括磷酸化和O-GlcNAc。此外，我们还研究了使用低能量Sori-CAD对O-GlcNAc和O-磷酸化肽进行碎裂和测序。在精心选择的条件下，Sori-CAD还可以导致肽骨架断裂，而修饰保持不变。O-GlcNAc修饰非常有趣，因为它发生在细胞核和细胞质中，似乎与磷酸化一样常见，并参与蛋白质信号转导。携带这种修饰的蛋白质，如RNA聚合酶II的C-末端结构域，表现出磷酸或O-GlcNAc修饰，这些修饰控制着蛋白质的活性状态(阴阳理论)。我们还利用LTQ-Orbitrap串联MS上的CID和C-陷阱解离以及Bruker四极离子陷阱和SolariX FTMS上的电子转移解离(ETD)研究了O-连接的葡聚糖。日本AIST的Narimatsu博士和他的同事正在提供合成参考标准。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Some O-linked protein modifications are reversible post-translational modifications of paramount importance in biological signaling pathways and are found in substoichiometric concentrations in the cell. This fact and the lability of these modifications under collisional activation conditions in the mass spectrometer make very difficult their detection by conventional tandem MS strategies. Several methods for detecting these modifications have been devised, including derivatization and labeling strategies. However, all these methods suffer from insufficient sensitivity. Derivatization methods using beta-elimination followed by Michael additions address this problem but introduce two new problems: i) phosphorylation and glycosylation compete for the same site and the conditions for beta-elimination are difficult to fine-tune such that only one modification is derivatized and not both; ii) derivatization methods remove the glycan and thus do not differentiate between complex O-glycosylation or single O-GlcNAc additions. In order to detect this extremely labile modification by MS, both the ionization methods and fragmentation methods used during analysis have to be extremely "soft" to keep the modification intact. Two instruments developed in this laboratory (vibrationally cooled MALDI FTMS and ESI-qQq FTMS) have proven to be ideal for both O-glycosylation and phosphorylation analysis as they both provide very soft ionization conditions and the ion optics are tunable such that the modified proteins/peptides are transferred the ICR cell intact. Electron Capture Dissociation (ECD) is the method of choice for inducing peptide fragmentation as it does not cleave the modifications from the peptide side chains. We have verified that ECD has the remarkable ability to extensively fragment peptides yielding almost complete sequence information (with the exception of proline) while not causing fragmentation within labile side-chain modifications including phosphorylation and O-GlcNAc. In addition, we have investigated the use of low-energy SORI-CAD for fragmenting and sequencing O-GlcNAc and O-phosphorylated peptides. Under carefully selected conditions, SORI-CAD can also result in peptide backbone fragmentation leaving the modification intact. The O-GlcNAc modification is extremely interesting as it occurs in the nucleus and cytoplasm, appears to be as common as phosphorylation, and is involved in protein signaling. Proteins that carry this modification, such as the C-terminal domain of RNA polymerase II exhibit either the phosphate or the O-GlcNAc modification, and these modifications govern the state of activity of the protein (Yin-Yang theory). We have also undertaken studies of O-linked glycans using CID and C-trap dissociation on the LTQ-Orbitrap tandem MS and Electron Transfer Dissociation (ETD) on the Bruker quadrupole ion traps and SolariX FTMS. Synthetic reference standards are being provided by Dr. Narimatsu and his colleagues at AIST, Japan.