TOPIC 319 TECHNOLOGY TO GENERATE ANTI PEPTIDE CAPTUE REAGENTS FOR AFFINITY ENRIFH

运营技术公司(Optech)建议针对NCI蛋白质组目标列表中的10个或更多磷酸化多肽开发DNA适配子。如果需要，这些适配子将被生物素化并偶联到链霉亲和素包裹的磁性微珠(MBS)上，或者共价结合到MBS上。适体-MB试剂将用于在杂交酶和胰酶处理的人血浆和血清中结合和丰富其特定的多肽。适体-MB试剂应直接转移到SISCAPA(稳定同位素标准和抗肽抗体捕获)等系统，但将具有以下优势：1)批次之间更好的重复性，因为适体DNA序列将被已知并以非常高的保真度复制；2)比可比的免疫磁性试剂更低的成本和更快的开发；3)与大多数抗体相比，潜在的更高的亲和力和特异性。适配子-MBS可以通过加热或酸化释放捕获的多肽，用于高通量蛋白质组系统的质谱分析。Optech还将试图通过在不同适配子末端化学连接不同的适配子来从血清或血浆中沉淀目标多肽，以开发能够连接多肽形成不可溶基质的双齿和三齿适配子。在第二阶段，Optech将改进和扩展这项技术，尽可能包括用于高通量蛋白质组学用途的多肽靶标的适体。

Operational Technologies Corp. (OpTech) proposes to develop DNA aptamers against ten or more phosphorylated peptides from NCI's list of proteomic targets. These aptamers will be biotinylated and coupled to streptavidin-coated magnetic microbeads (MBs) or covalently bound to MBs, if necessary. The aptamer-MB reagents will be used to bind and enrich for their specific peptides in chaotrope and trypsin-treated human plasma and serum. Aptamer-MB reagents should be directly transferred to systems such as SISCAPA (Stable Isotope Standards and Capture by Anti-Peptide Antibodies), but will have the advantage of: 1) grater lot-to-lot reproductivity because aptamer DNA sequences will be known and reproduced with very high fidelity, 2) lower cost and faster development than comparable immunomagnetic reagents, 3) potentially greater affinity and specificity versus most antibodies. Aptamer-MBs can then release captured peptides by heating or acidification for mass spectral analyses in high throughput proteomic systems. OpTech will also attempt to precipitate target peptides from serum or plasma by chemically linking different aptamers at their ends to develop bi- and tridentate aptamers capable of linking peptides to form insoluble matrices. In Phase 2, OpTech will refine and expand the technology to include as may aptamers for peptide targets as possible for high-throughput proteomic use.