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

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 一些O-连接的蛋白质修饰是可逆的翻译后修饰，在生物信号传导途径中至关重要，并且在细胞中以亚化学计量浓度存在。这一事实和在质谱仪中碰撞活化条件下这些修饰的不稳定性使得它们很难通过常规串联MS策略检测。已经设计了几种用于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-消除接着迈克尔加成的衍生化方法解决了这个问题，但引入了两个新问题：i）磷酸化和糖基化竞争相同位点，并且β-消除的条件难以微调，使得仅一种修饰被衍生化，而不是两种修饰都被衍生化; ii）衍生化方法去除聚糖，因此不能区分复杂的O-糖基化或单一的O-GlcNAc加成。为了通过MS检测这种极其不稳定的修饰，在分析期间使用的电离方法和裂解方法都必须非常“软”以保持修饰完整。该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选择，因为它们都提供了非常软的电离条件，并且离子光学器件是可调的，因此修饰的蛋白质/肽可以完整地转移到ICR细胞中。电子捕获解离（ECD）是诱导肽片段化的首选方法，因为它不会从肽侧链切割修饰。我们已经证实，ECD具有显著的能力，广泛片段肽产生几乎完整的序列信息（除脯氨酸），而不引起不稳定的侧链修饰，包括磷酸化和O-GlcNAc内的片段。此外，我们还研究了使用低能量SORI-CAD对O-GlcNAc和O-磷酸化肽进行片段化和测序。在精心选择的条件下，SORI-CAD还可以导致肽骨架断裂，使修饰保持完整。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，并且参与蛋白质信号传导。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，这些修饰控制蛋白质的活性状态（阴阳理论）。我们还在LTQ-Orbitrap串联MS上使用CID和C阱解离以及在Bruker四极离子阱和SolariX FTMS上使用电子转移解离（ETD）对O-连接聚糖进行了研究。合成参比标准品由日本AIST的Narimatsu博士及其同事提供。

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.