Affinity Capture Peptides for Clinical Mass Spectrometric Assays in Plasma

描述(由申请人提供)：我们将为所有14种骨形态发生蛋白的多重组合开发第一种分析方法。免疫分析是测量临床样本中蛋白质的“金标准”。然而，这些基于抗体的检测既不能量化蛋白质分析物的变异体，也不能在大量的超家族成员中进行多重检测。此外，多克隆和单克隆抗体的可用性也是有限的。我们针对这些问题提出了一种新的解决方案，首先通过修改质谱学免疫捕获分析方法，使用亲和捕获多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高效、高通量生产合成肽作为靶向捕获剂。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个骨形态发生蛋白(BMP)成员的多重组合。第二阶段将验证整个BMP/转化生长因子的多重分析-？(转化生长因子？)超级家族，其33个成员在发育、组织维护和许多疾病中发挥着关键作用。转化生长因子-？与诊断和治疗相关的超家族成员包括Myostatin(抑制增加肌肉和骨骼，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过高表达)，BMP2和4(在退行性关节疾病中过表达)，TGFs-1，2和3(复杂的癌症参与)，以及BMP2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、具体和快速的检测，以检测这个庞大而重要的家族中的单个成员或选定的成员组合。

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.