APPLICATION OF IN-SOURCE DECAY IN ISOASPARTOMICS RESEARCH

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。 已知ECD可生成isoAsp特异性诊断离子，用于isoAsp鉴定和定量。还使用纳米液相色谱（nanoLC）-ECD方法证明了蛋白质组规模的isoAsp作图。然而，ECD仅对通常由电喷雾电离（ESI）产生的多电荷阳离子起作用。一些肽不能被ESI有效地电离，特别是在存在电离抑制剂的情况下。此外，给定肽的洗脱时间可能太短而不能允许足够的信号平均以用于诊断离子的观察。最后，离线LC-ESI-ECD分析通常需要皮摩尔的样品，这并不总是可用于临床来源的样品。 基质辅助激光解吸/离子化（MALDI）提供了一种互补的离子化方法，可用于提高蛋白质覆盖率，当样品量有限时。与ESI相比，MALDI对电离抑制的敏感性更低。作为离线技术，对于MALDI生成的离子的分析，信号平均没有时间限制。使用MALDI也可以进行飞摩尔样品的串联MS分析。然而，MADLI主要产生单电荷离子，由于其电荷还原性质，其不能通过ECD分析。我们目前正在探索MALDI产生的离子的isoAsp分析的替代方法。一种这样的方法是源内衰变（ISD），其主要产生c-和z-型离子，推测由氢自由基从基质转移到分析物离子引发。初步数据显示ISD在Bruker Reflex IV MALDI-TOF仪器上工作良好。在合成的含isoAsp肽的ISD光谱中也观察到质量与诊断离子的质量相对应的峰，尽管它们位于基质簇干扰峰丰富的光谱区域（<1000 Da）。我们目前正在探索减少基质簇干扰的方法。

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. ECD is known to generate isoAsp-specific diagnostic ions for isoAsp identification and quantitation. Proteome-scale isoAsp mapping has also been demonstrated using a nano liquid chromatography (nanoLC)-ECD method. However, ECD only works with multiply-charged cations that are typically generated by electrospray ionization (ESI). Some peptides are not efficiently ionized by ESI, particularly in the presence of ionization suppression agents. Further, the elution time of a given peptide may be too short to allow sufficient signal averaging for the observation of diagnostic ions. Finally, offline LC-ESI-ECD analysis typically requires picomoles of sample, which are not always available for samples of clinical origin. Matrix-assisted laser desorption/ionization (MALDI) offers a complementary ionization method that can be used to improve the protein coverage and when sample amount is limited. Comparing with ESI, MALDI is less susceptible to ionization suppression. As an offline technique, there is no time constraint for signal averaging for analyses of MALDI-generated ions. Tandem MS analysis with femtomoles of sample is also possible with MALDI. However, MADLI produces predominantly singly-charged ions, which cannot be analyzed by ECD due to its charge-reducing nature. We are currently exploring alternative methods for isoAsp analysis of MALDI-generated ions. One such method is in-source decay (ISD), which produces predominantly c- and z-type ions, presumably initiated by hydrogen radical transfer from the matrix to the analyte ions. Preliminary data shows ISD works well on the Bruker Reflex IV MALDI-TOF instrument. Peaks with mass corresponding to those of diagnostic ions were also observed in the ISD spectrum of a synthetic isoAsp-containing peptide, although they were located at a spectral region (<1000 Da) where interference peaks of matrix clusters abound. We are currently exploring ways to reduce the matrix cluster interference.