SAMPLE PREPARATION; The Achilles Heel of Mass Spectrometry Based Diagnostics II.

样品制备;

• 批准号: 9409052
• 负责人: Jinhee Kim
• 金额: $ 74.96万
• 依托单位: NOVILYTIC, LLC
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-03-01 至 2019-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9409052
• 关键词: Affinity; Affinity Chromatography; Aliquot; Antibodies; Biological Assay; Biological Markers; Biological Sciences; Blood; C-reactive protein; Cells; Chromatography; Clinical Research; Collection; Complex; Cost Analysis; Coupled; Data; Detection; Diagnostic; Dissociation; Drops; Excision; Fingers; Fluorescence; Funding; GTP-Binding Protein alpha Subunits, Gs; Goals; Immunoassay; Immunology procedure; In Situ; Laboratories; Mass Spectrum Analysis; Medicine; Membrane; Methods; Molecular Conformation; Performance; Phase; Plasma; Post-Translational Protein Processing; Preparation; Protein Analysis; Proteins; Protocols documentation; Recovery; Research; Resolution; Route; Sampling; Series; Solvents; Spectrometry, Mass, Electrospray Ionization; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Structure; Surface; System; Techniques; Technology; Time; Variant; base; clinical diagnostics; cost; detector; drug testing; innovation; instrument; instrumentation; interest; ionization; nanoparticulate; particle; reversed phase chromatography; sample collection; targeted biomarker; tool

SAMPLE PREPARATION; The Achilles Heel of Mass Spectrometry Based Diagnostics II. Project Summary. LC-MS provides superior data in clinical diagnostics relative to immunoassays, but it is slow and costly. As the old saying goes, time is money. The technique must become simpler, faster, and less expensive to displace older, less accurate immunological assay (IA) methods. The focus of this proposal is to make LC-MS analytics more competitive in clinical diagnostics by increasing throughput 10 fold with existing instrumentation. The objective in Phase I was to use analyte sequestering transport particles (ASTPs) to circumvent the above limitations by i) structure specifically sequestering analytes of interest, ii) separating them from non-analytes within a min, ii) rapidly transferring them to an MS, and iv) enabling the MS to identify all analytes of interest in less than 60 sec. This was achieved by preparing 2 megadalton (mDa) ASTPs that sequester analytes at their surface; elevating their effective Mw to 2 mDa and allowing them to be purified by mobile affinity sorbent chromatography (MASC) in 60 sec. Thermal or solvent dissociation was used to recover analytes for ESI-MS or LDTD-MS wells. This proposal has four specific aims. One is to complete manufacturing protocols for antibody and protein A/G based ASTP products that capture, resolve, and transport analytes to MS or fluorescence instruments in research, big pharma, and CRO laboratories. The second is to integrate the above ASTP products into miniature membrane laboratories (MemLabs); producing point-of-collection products that extract plasma from a drop of blood and prepare samples in situ for biomarker detection. The third aim was to evaluate and optimize the release and detection of analytes from ASTPs via LDTD-MS, MALDI-MS, and ESI-MS. The fourth specific aim is to adapt ASTP technology to multiplexed drug testing and the analysis of protein i) primary structure, ii) post-translational modifications, and iii) conformation.

样品制备；基于质谱的诊断的致命弱点 II。 项目摘要。 相对于免疫分析，LC-MS 在临床诊断方面提供了更优质的数据，但其速度慢且 昂贵。俗话说，时间就是金钱。该技术必须变得更简单、更快、更少 取代旧的、不太准确的免疫学测定 (IA) 方法的成本很高。本次的重点 提议是通过提高通量来使 LC-MS 分析在临床诊断中更具竞争力 是现有仪器的 10 倍。 第一阶段的目标是使用分析物隔离运输颗粒 (ASTP) 来规避 上述限制通过 i) 结构专门隔离感兴趣的分析物，ii) 分离它们 一分钟内从非分析物中分离出来，ii) 快速将其转移至 MS，以及 iv) 使 MS 能够 在 60 秒内识别所有感兴趣的分析物。这是通过准备 2 兆道尔顿 (mDa) 来实现的 ASTP 将分析物隔离在其表面；将它们的有效分子量提高到 2 mDa，并允许它们 通过移动亲和吸附色谱 (MASC) 在 60 秒内纯化。热或溶剂 解离用于回收 ESI-MS 或 LDTD-MS 孔的分析物。 该提案有四个具体目标。一是完成抗体和 基于 Protein A/G 的 ASTP 产品，可捕获、解析分析物并将其传输至 MS 或荧光 研究、大型制药公司和 CRO 实验室的仪器。二是整合以上内容 ASTP产品进入微型膜实验室（MemLabs）；生产收集点 从一滴血中提取血浆并原位制备样品以进行生物标志物检测的产品。 第三个目标是通过 ASTP 评估和优化分析物的释放和检测 LDTD-MS、MALDI-MS 和 ESI-MS。第四个具体目标是使 ASTP 技术适应 多重药物测试和蛋白质分析 i) 一级结构，ii) 翻译后 修饰，以及 iii) 构象。