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

样品制备；基于质谱学的阿喀琉斯跟踪法2。 项目摘要。 与免疫分析相比，LC-MS在临床诊断中提供了更好的数据，但它速度慢， 代价不菲。正如老话所说，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、不太准确的免疫分析(IA)方法成本高昂。这件事的重点是 建议通过增加吞吐量使LC-MS分析在临床诊断中更具竞争力 10倍与现有的仪器。 第一阶段的目标是使用分析物隔离传输粒子(ASTP)来绕过 上述限制由i)结构具体隔离感兴趣的分析物，ii)分离它们 在一分钟内从非分析物中分离出来，ii)将它们快速转移到MS，以及iv)使MS能够 在不到60秒的时间内确定所有感兴趣的分析物。这是通过准备200万吨丙二醛(MDA)实现的 将分析物隔离在其表面的ASTP；将其有效分子量提高到2mda，并允许它们 用流动亲和层析(MASC)在60秒内纯化。热或溶剂 解离用于回收ESI-MS或LDTD-MS井中的分析物。 这项提议有四个具体目标。一是完成抗体和抗体的制造方案 基于蛋白A/G的ASTP产品，捕获、解析分析物质并将其传输到MS或荧光 研究仪器、大型制药公司和CRO实验室。二是整合上述内容 ASTP产品进入微型膜实验室(MemLabs)；生产采集点 从一滴血中提取血浆并在现场准备样品以进行生物标志物检测的产品。 第三个目标是评估和优化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 多重药物检测和蛋白质分析--I)一级结构，II)翻译后 修饰，以及iii)构象。

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.