Mass Spectrometry based molecular imaging of native biological nanodomains

描述(由申请者提供)：申请书建议弗朗西斯科·A·费尔南德斯·利马博士的职业发展，他是一名博士后研究员，受过生物质谱学和仪器与方法开发方面的培训。费尔南德斯-利马博士致力于生物物理科学的研究生涯，通过创造新的和改进现有的技术和方法，在分子和细胞水平上研究科学问题，这些技术和方法可以进一步转化为基于动物的研究，以患者为导向的临床研究，并最终改善患者护理。申请者将由Emile A.Schweikert博士指导用于质谱学的纳米级成像探针，由David H.Russell博士在生物质谱学仪器和方法开发方面联合指导，并由Jennifer L.Bizon博士在行为和细胞神经科学方法以及认知障碍疾病研究的动物模型方面共同指导。该项目将在德克萨斯A&M大学进行，提议用仪器开发一个质谱仪，与一个纳米成像探头相连，能够在单细胞和亚细胞水平上询问自然生物表面(目前在提议的水平上尚不具备)。该仪器(特定目标1)将使用高达100 qkeV能量的簇束探测器(Au100n+Q和Binq+Q)，以增强分子产额发射(~10倍增加)，并使用电子发射显微镜进行横向分辨率低于100 nm的分子离子定位。该方法将使用包含已知表面标记(例如，免疫细胞(Molt-3)和肝细胞的CD4抗原和肝细胞生长因子受体(c-MET)的表达)的明确定义的细胞系统来验证，以表征仪器性能(特定目标2)。快速气相分离(在这种情况下，离子迁移率-质谱仪，IM-MS)和裂解技术(IM-CID-MS)将被应用于分子生物标志物的分离和鉴定(具体目标3)。作为一个短期目标，神经元的表型表达、形态和/或稳定性将与具有行为特征的年轻、中年和老年F344大鼠的基底前脑化学环境相关(特定目标4)。相关性：该项目将通过使用未经处理的组织样本直接将化学环境与其生物功能相关联，为与认知障碍疾病相关的分子标记的单细胞和亚细胞研究奠定工具和方法基础。 公共卫生相关性：细胞水平上的生物学表现受化学环境和表面化学的调节。一种新的仪器和方法将被创造出来，它可以检测天然生物表面的分子组成。一个独特的功能将是生物标记的定位，分辨率将比光学显微镜提高100倍。

DESCRIPTION (provided by applicant): The application proposes a career development for Dr. Francisco A. Fernandez-Lima, a postdoctoral fellow trained in biological mass spectrometry and instrument & method development. Dr. Fernandez- Lima is committed to a research career in biophysical science to study scientific problems at a molecular and cellular levels by creating new and improving current techniques and methods that can be further translated to animal based studies, to patient-oriented clinical research, and ultimately lead to improved patient care. The applicant will be mentored by Dr. Emile A. Schweikert in nanometer scale imaging probes for mass spectrometry, co-mentored by Dr. David H. Russell in instrument and method development for biological mass spectrometry, and co-mentored by Dr. Jennifer L. Bizon in behavioral and cellular neuroscience methods and animal models for studies of cognitive impairment diseases. The project, to be conducted at Texas A&M University, proposes the instrumental development of a mass spectrometer coupled to a nanometer imaging probe capable of interrogating native biological surfaces at the single cell and sub-cellular levels (currently not available at the level proposed). The instrument (Specific Aim 1) will employ a cluster beam probe (Au100n+q and Binq+q) at up to 100 qkeV energies for enhanced molecular yield emission (~10 fold increase), and molecular ion localization with sub-100nm lateral resolution using an electron emission microscope. The methodology will be validated using well-defined cellular systems containing known surface markers (e.g., expression of CD4 antigen and hepatocyte growth factor receptor (c-met) from Immune cells (Molt-3) and hepatocytes) to characterize the instrument performance (Specific Aim 2). Fast gas-phase separation (in this case Ion Mobility - Mass Spectrometry, IM-MS) and fragmentation techniques (IM-CID-MS) will be applied to the separation and identification of molecular biomarkers (Specific Aim 3). As a short- term goal, the neuron phenotypic expression, morphology, and/or stability will be correlated with the basal forebrain chemical environment of behaviorally characterized young, middle-aged, and aged F344 rats (Specific Aim 4). Relevance: The project will set the instrumental and methodological basis for single cell and sub-cellular studies of molecular markers associated with cognitive impairment diseases by directly correlating the chemical environment with their biological function using untreated tissue samples. PUBLIC HEALTH RELEVANCE: The biological performance at the cellular level is mediated by the chemical environment and surface chemistry. A new instrument and method will be created which can examine molecular composition on native biological surfaces. A unique feature will be the localization of biological markers with a resolution improved one hundred-fold over light microscopy.