Affinity Capture Peptides for Clinical Mass Spectrometric Assays in Plasma

描述（由申请人提供）：我们将开发所有14种骨形态发生蛋白的多重组合的第一个检测。免疫测定法是测量临床样品中蛋白质的“金标准”。然而，这些基于抗体的测试既不能定量蛋白质分析物变异，也不能在大量超家族成员中多重分布。此外，多克隆和单克隆抗体的可用性是有限的。针对这些问题，我们提出了一种新的解决方案，首先通过修改质谱免疫捕获测定，使用亲和捕获肽来丰富临床相关的蛋白质变体。其次，利用噬菌体展示文库中高效、高通量的合成多肽作为靶向捕获试剂。为了概念验证，我们将开发一种定量原型分析，用于人类血浆中所有14种骨形态发生蛋白（BMP）成员的多路组合。II期将验证整个BMP/转化生长因子-？（TGF-?）超家族，其33个成员在发育、组织维持和许多疾病中起着至关重要的作用。TGF - ?与诊断和治疗应用相关的超家族成员包括肌生长抑制素（抑制增加肌肉和骨骼并减少脂肪）、BMP-2（在肺癌、胃癌和胶质瘤中过表达）、BMP-4（在慢性肾脏疾病和冠状动脉疾病中升高）、BMP-2和4（在退行性关节疾病中过表达）、tgf -？BMPs 2和7（被批准用于骨不连骨折和脊柱融合）。这些新的多重检测方法将为研发、诊断和治疗市场提供可靠、特异性和更快的检测这一重要大家族成员的单一或选择组合。

DESCRIPTION (provided by applicant): We will develop the first assay for multiplexed combinations of all 14 Bone Morphogenetic Proteins. Immunoassays are the "gold standard" for measuring proteins in clinical samples. However these antibody- based tests neither quantitate protein analyte variants, nor multiplex across large numbers of superfamily members. In addition, the availability of polyclonal and monoclonal antibodies is limited. We propose a novel solution to these problems, firstly by modifying a mass spectrometric immunocapture assay to use affinity capture-peptides to enrich clinically relevant protein variants. Secondly by exploitin the efficient and high- throughput production of synthetic peptides from phage display libraries as targeted capture-reagents. For proof-of-concept, we will develop a quantitative prototype assay for multiplexed combinations of all 14 Bone Morphogenetic Protein (BMP) members in human plasma. Phase II will validate multiplexed assays for the whole BMP/Transforming Growth Factor-? (TGF-?) superfamily, whose 33 members play crucial roles in development, tissue maintenance, and many diseases. TGF-? superfamily members relevant to diagnostic and therapeutic applications include Myostatin (inhibition increases muscle and bone and decreases fat), BMP-2 (overexpressed in lung carcinomas, gastric carcinomas, and gliomas), BMP-4 (elevated in chronic kidney disease and coronary artery disease), BMPs 2 and 4 (overexpressed in degenerative joint disease), TGFs-? 1, 2 and 3 (complex involvement in cancer), and BMPs 2 and 7 (approved for nonunion bone fractures and spinal fusions). These new multiplexed assays will enable reliable, specific and faster detection of single or selected combinations of members of this large and important family, for the R&D, diagnostics and therapeutics markets.