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

基本信息

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
项目类别：
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

项目摘要

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）：蛋白质组分析需要对复杂生物混合物中的蛋白质进行鉴定和定量。在目前的蛋白质组分析方法中，越来越多地应用“鸟枪法”分析，其中蛋白质混合物在开始时被消化以产生甚至更复杂的片段肽混合物。片段肽的分析；它与前体蛋白一样，可以以广泛变化的浓度存在，提出了重大的分析挑战。为了观察更多的肽混合物成分，特别是通常预期具有最大功能意义的低丰度蛋白质片段，需要改进的方法来分离肽（增加“分离空间”）和观察更多的肽。给定的分离分数（增加“分析深度”）。电喷雾电离 (ESI) 和基质辅助激光解吸电离 (MALDI) 质谱 (MS) 是蛋白质组分析中使用的主要方法，倾向于观察复杂混合物中肽的互补子集。两种方法的应用比单独使用任何一种方法都具有更大的覆盖范围，但这些方法通常需要在不同的 MS 仪器上使用不同的样品格式，因此需要更多的样品以及更多的时间、精力和费用来应用这两种方法。这项拟议的工作旨在通过使用采用纳米结构表面的新平台来增加肽分析深度，从而能够对同一样品应用互补电离模式、激光解吸电离 (LDI) 和解吸电喷雾电离 (DESI)。在目标 1 中，我们将优化纳米结构氧化铝 (Al2O3) 表面在肽混合物 LC 分离组分的 LDI 和 DESI 分析中的使用。在目标 2 中，我们将开发一种改进的 DESI 源，以促进密集样品点阵列的双模式分析。由此产生的平台将增加分析深度，并通过增加馏分收集密度来促进增加分离空间。完全离线的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的不一致；提高MS仪器时间利用效率；并为以后的重新分析提供可存档的介质。因此，这项工作将显着推进可用于更完整地分析蛋白质组样品的可用技术。公共卫生相关性：蛋白质组学研究有望产生用于疾病诊断和预防的新生物标志物，并确定开发改进疗法和疾病预防方法的新目标。拟议的工作将开发普遍适用的新分析方法，从而提高生物医学研究广泛领域（包括心血管疾病、癌症和心理健康等）蛋白质组学研究的信息产量。