APPLICATION OF IN-SOURCE DECAY IN ISOASPARTOMICS RESEARCH

源内衰变在等天组学研究中的应用

• 批准号: 8365576
• 负责人: CHI-WEI LIN
• 金额: $ 0.31万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-06-01 至 2012-08-09
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8365576
• 关键词: Biology; Cations; Charge; Clinical; Data; Diagnostic; Electrospray Ionization; Funding; Grant; Hydrogen; Ions; Liquid Chromatography; Maps; Mass Spectrum Analysis; Medicine; Methods; National Center for Research Resources; Nature; Peptides; Principal Investigator; Proteins; Proteome; Reflex action; Research; Research Infrastructure; Resources; SHFM1 gene; Sampling; Signal Transduction; Source; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Techniques; Time; United States National Institutes of Health; Work; cost; improved; instrument; ionization; ionization technique; nano

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.

该子项目是利用资源的众多研究子项目之一 由 NIH/NCRR 资助的中心拨款提供。子项目的主要支持 并且子项目的主要研究者可能是由其他来源提供的， 包括其他 NIH 来源。 子项目可能列出的总成本 代表子项目使用的中心基础设施的估计数量， NCRR 赠款不直接向子项目或子项目工作人员提供资金。 ECD 已知可产生 isoAsp 特异性诊断离子，用于 isoAsp 识别和定量。蛋白质组规模的 isoAsp 图谱也已使用纳米液相色谱 (nanoLC)-ECD 方法得到了证明。然而，ECD 仅适用于通常由电喷雾电离 (ESI) 产生的多电荷阳离子。有些肽不能被 ESI 有效电离，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无法对诊断离子的观察进行足够的信号平均。最后，离线 LC-ESI-ECD 分析通常需要皮摩尔样品，这并不总是适用于临床来源的样品。 基质辅助激光解吸/电离 (MALDI) 提供了一种补充电离方法，可用于在样品量有限时提高蛋白质覆盖率。与 ESI 相比，MALDI 不易受到电离抑制。作为一种离线技术，MALDI 生成离子分析的信号平均没有时间限制。 MALDI 还可以对样品进行串联 MS 分析。然而，MADLI 主要产生单电荷离子，由于其电荷还原性质，无法通过 ECD 进行分析。我们目前正在探索 MALDI 生成离子的 isoAsp 分析的替代方法。其中一种方法是源内衰变 (ISD)，它主要产生 c 型和 z 型离子，可能是由氢自由基从基质转移到分析物离子引发的。初步数据显示 ISD 在 Bruker Reflex IV MALDI-TOF 仪器上运行良好。在合成的含 isoAsp 的肽的 ISD 光谱中也观察到了与诊断离子质量相对应的峰，尽管它们位于基质簇干扰峰大量存在的光谱区域 (<1000 Da)。我们目前正在探索减少矩阵簇干扰的方法。