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;质谱诊断的致命弱点2。 项目摘要。 相对于免疫测定，LC-MS在临床诊断中提供了上级数据，但其缓慢且 很贵。正如那句老话所说，时间就是金钱。这项技术必须变得更简单、更快、更少 取代较旧的、不太准确的免疫测定（IA）方法是昂贵的。的重点 一项提案是通过提高通量，使LC-MS分析在临床诊断中更具竞争力 10倍于现有仪器。 第一阶段的目标是使用分析物螯合转运颗粒（ASTP）来规避 通过i）结构特异性地螯合感兴趣分析物，ii）分离它们 ii）将它们快速转移到MS，以及iv）使MS能够 在不到60秒内鉴定所有感兴趣分析物。这是通过制备2兆道尔顿（mDa） 在其表面螯合分析物的ASTP;将其有效Mw提高到2 mDa，并允许它们 在60秒内通过移动的亲和吸附层析（MASC）纯化。热或溶剂 解离用于回收ESI-MS或LDTD-MS威尔斯孔的分析物。 这项建议有四个具体目标。一个是完成抗体的生产方案， 基于蛋白A/G的ASTP产品，可捕获、解析分析物并将其传输至MS或荧光分析仪 研究、大型制药和CRO实验室的仪器。二是整合上述 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用于生物标志物检测的产品。 第三个目的是通过以下方法评估和优化ASTP中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技术适应于 多重药物检测和蛋白质分析i）一级结构，ii）翻译后 修饰，和iii）构象。