Dual Mode Mass Spectrometry Platform for Increasing Depth in Proteomic Analysis

用于增加蛋白质组分析深度的双模式质谱平台

• 批准号: 7941076
• 负责人: DANIEL R. KNAPP
• 金额: $ 25.55万
• 依托单位: MEDICAL UNIVERSITY OF SOUTH CAROLINA
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2013-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7941076
• 关键词: Address; Aluminum Oxide; Archives; Area; Arts; Biological; Biological Markers; Biomedical Research; Cardiovascular Diseases; Complex; Complex Mixtures; Crystallization; Data; Development; Diagnosis; Electrospray Ionization; Goals; Human; Iceberg; Image; Ions; Lasers; Malignant Neoplasms; Mass Spectrum Analysis; Mental Health; Methodology; Methods; Names; Peptide Fragments; Peptides; Performance; Phase; Preparation; Prevention approach; Protein Precursors; Proteins; Proteome; Proteomics; Public Health; Research; Resolution; Running; Sampling; Shotguns; Source; Spectrometry, Mass, Electrospray Ionization; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Spottings; Surface; Technology; Time; Tissues; Vacuum; Work; analytical method; density; disorder prevention; improved; innovation; instrument; ion source; ionization; nanostructured; novel strategies; public health relevance; Address; Aluminum Oxide; Archives; Area; Arts; Biological; Biological Markers; Biomedical Research; Cardiovascular Diseases; Complex; Complex Mixtures; Crystallization; Data; Development; Diagnosis; Electrospray Ionization; Goals; Human; Iceberg; Image; Ions; Lasers; Malignant Neoplasms; Mass Spectrum Analysis; Mental Health; Methodology; Methods; Names; Peptide Fragments; Peptides; Performance; Phase; Preparation; Prevention approach; Protein Precursors; Proteins; Proteome; Proteomics; Public Health; Research; Resolution; Running; Sampling; Shotguns; Source; Spectrometry, Mass, Electrospray Ionization; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Spottings; Surface; Technology; Time; Tissues; Vacuum; Work; analytical method; density; disorder prevention; improved; innovation; instrument; ion source; ionization; nanostructured; novel strategies; public health relevance

DESCRIPTION (provided by applicant): Proteomic analysis entails identification and quantitation of the proteins in complex biological mixtures. Of the present proteomic analysis methods, "shotgun" analysis, wherein the protein mixture is digested at the outset to yield an even more complex mixture of fragment peptides, is being increasingly applied. Analysis of the fragment peptides; which, like the precursor proteins, can be present in widely varying concentrations, presents a significant analytical challenge. To observe more of the peptide mixture components, particularly the fragments of low abundance proteins which are generally expected to be of greatest functional significance, requires improved methods both for separating the peptides (increasing "separation space") and for observing more of the peptides in a given separated fraction (increasing "analysis depth"). Electrospray ionization (ESI) and matrix assisted laser desorption ionization (MALDI) mass spectrometry (MS), the primary methods used in proteomic analysis, tend to observe complementary subsets of the peptides in complex mixtures. Application of both methods gives greater coverage than either alone, but the methods entail different sample formats usually on different MS instruments and thus require more sample and more time, effort, and expense to apply both. This proposed work is directed toward increasing peptide analysis depth via use of a new platform employing nanostructured surfaces to enable application of complementary ionization modes, laser desorption ionization (LDI) and desorption electrospray ionization (DESI), to the same sample. In Aim 1, we will optimize the use of nanostructured alumina (Al2O3) surfaces for LDI and DESI analysis of peptide mixture LC separation fractions. In Aim 2, we will develop an improved DESI source to facilitate dual mode analysis of dense arrays of sample spots. The resulting platform will yield increased analysis depth as well as facilitate increasing separation space by enabling increased density of fraction collecting. The totally offline sample preparation will remove the time constraints of online analysis, which can produce inconsistency in peptide observations; improve efficiency of MS instrument time utilization; and provide an archivable medium for later reanalysis. As such, the work will significantly advance the available technologies for more complete analysis of proteomic samples. PUBLIC HEALTH RELEVANCE: Proteomics studies promise to yield new biomarkers for diagnosis and prevention of disease as well as identify new targets for development of improved therapies and disease prevention approaches. The proposed work will develop generally applicable new analytical methods that will increase the information yield from proteomics studies in wide-ranging areas of biomedical research including cardiovascular disease, cancer, and mental health to name a few.

描述（由申请人提供）：蛋白质组学分析需要在复杂的生物混合物中鉴定和定量蛋白质。在当前的蛋白质组学分析方法中，“ shot弹枪”分析（一开始就消化蛋白质混合物，以产生片段肽的更复杂的混合物，正在越来越多地应用。分析碎片肽；像前体蛋白一样，它可以以广泛变化的浓度存在，提出了重大的分析挑战。要观察更多的肽混合物成分，尤其是通常预期具有最大功能意义的低丰度蛋白质的片段，需要改进的方法来分离肽（增加“分离空间”），以及在给定的分离馏分中观察更多的肽（增加“分析”分析“分析”）。电喷雾电离（ESI）和基质辅助激光解吸电离（MALDI）质谱法（MS）是蛋白质组学分析中使用的主要方法，倾向于在复杂混合物中观察肽的互补子集。两种方法的应用都比任何一个单独的都提供了更大的覆盖范围，但是通常在不同的MS仪器上需要不同的样本格式，因此需要更多的样本以及更多的时间，精力和费用才能应用两者。这项提出的工作是针对通过使用纳米结构表面的新平台来增加肽分析深度的，以实现互补电离模式，激光解吸电离（LDI）和解吸电喷雾电离（DESI），以在同一样本中。在AIM 1中，我们将优化使用纳米结构氧化铝（Al2O3）表面进行LDI和DESI分析的肽混合物LC分离分数。在AIM 2中，我们将开发一个改进的DESI源，以促进样品斑点密集阵列的双重模式分析。所得平台将通过增加分数收集密度来增加分析深度，并促进分离空间的增加。完全离线样品制备将消除在线分析的时间限制，这可能会导致肽观察结果不一致；提高MS仪器时间利用的效率；并提供可存档的介质，以供以后重新分析。因此，这项工作将大大推进可用技术，以对蛋白质组学样品进行更完整的分析。 公共卫生相关性：蛋白质组学研究有望产生新的生物标志物来诊断和预防疾病，并确定开发改进的疗法和预防疾病方法的新目标。拟议的工作将开发通常适用的新分析方法，该方法将增加生物医学研究广泛领域的蛋白质组学研究的信息产量，包括心血管疾病，癌症和心理健康。