Quantifying proteins in plasma do democratize personalized medicine for patients with type 1 diabetes

摘要 1型糖尿病在美国影响着超过125万人， 发病率正以惊人的3- 4%的速度增长。的情感和经济负担， 疾病是压倒性的，我们目前没有办法预测或预防新的病例。正如我们 更好地了解胰腺的病理生理过程， 高血糖的下游效应（以及治疗期间的周期性低血糖），更多 需要稳健的生物标志物测定来提高研究结果的再现性， 将这些发现转化为临床护理。一种技术，可以提供强大的，可转移的 用于测量蛋白质的测定法是液相色谱-串联质谱法。 通过直接检测感兴趣的分析物，使用质谱检测的测定可以 具有比免疫测定更好的特异性，当与富集策略配对时， 也可能非常敏感。正如我们之前所展示的， 协调质谱分析的结果，这对于 一般的免疫测定。该提案旨在生成和验证新颖的可转让资产 利用质谱分析的力量进行蛋白质分析。我们的目标是利用一种新的方法 为了富集细胞外囊泡和用于亲和富集的新的从头蛋白质， 叫做微型结合物，以提高方法的灵敏度。只要有可能， 多路复用，如果需要抗体进行富集，它们将通过 爱荷华州混合银行编码小结合物的质粒将保藏在Addgene。 方法开发的色谱数据（特别是肽选择，将使用 窄窗口数据独立采集，而不是依赖于算法或数据， 依赖的采集方法）以及方法验证的色谱数据， 沿着详细的标准作业程序。所要求 RFA DK-21-031，所产生的测定的一部分将靶向胰高血糖素， 胰高血糖素原、胰岛素原及其片段、糖化可溶性CD 59、胰淀素和胰高血糖素原。 嗜铬粒蛋白我们的目标优先级委员会将帮助确定最重要的 蛋白质添加到这个列表中，并集中我们的发展努力。

ABSTRACT Type 1 diabetes affects more than 1.25 million people in the United States and the annual incidence is increasing at an alarming rate of 3-4%. The emotional and financial burden of the disease is overwhelming and we currently have no way to predict or prevent new cases. As we gain a better understanding of the pathophysiological processes in the pancreas and the downstream effects of hyperglycemia (and periodic hypoglycemia during treatment), more robust biomarker assays are needed to improve the reproducibility of research findings and to translate those findings to clinical care. One technology that can provide robust, transferable assays for the measurement of proteins is liquid chromatography-tandem mass spectrometry. By directly detecting the analyte of interest, assays that use mass spectrometry detection can have better specificity than immunoassays and when paired with enrichment strategies, they can also be very sensitive. As we have demonstrated previously, it is straightforward to harmonize the results of mass spectrometric assays, which is significantly more difficult for immunoassays in general. This proposal aims to generate and validate novel transferable protein assays that harness the power of mass spectrometry. We aim to leverage a new method for the enrichment of extracellular vesicles and new de novo proteins for affinity enrichment, called minibinders, to help with sensitivity of the methods. Whenever possible, assays will be multiplexed and if antibodies are required for enrichment, they will be widely distributed through the Iowa Hybridoma Bank. Plasmids encoding minibinders will be deposited at Addgene. Chromatographic data from method development (particularly peptide selection, which will use narrow-window data-independent acquisition rather than relying on algorithms or data- dependent acquisition methods) as well as chromatographic data from method validation will be distributed via Panorama, along with detailed standard operating procedures. As requested in RFA DK-21-031, a portion of the assays produced will target glucagon, other fragments of proglucagon, proinsulin and its fragments, glycated soluble CD59, amylin, and the chromogranins. Our Target Prioritization Committee will help identify the most important proteins to add to this list and focus our development efforts.