A comprehensive cardiac troponin assay enabled by nanotechnology and top-down proteomics

通过纳米技术和自上而下的蛋白质组学实现全面的心肌肌钙蛋白测定

• 批准号: 8909604
• 负责人: Tania Maria Guardado
• 金额: $ 4.06万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-15 至 2016-06-25
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8909604
• 关键词: Acute myocardial infarction; Affinity; Antibodies; Area; Binding; Biological Assay; Biological Markers; Blood; Blood specimen; Cardiac; Cardiac Myocytes; Cataloging; Catalogs; Cause of Death; Characteristics; Chemistry; Complex; Complex Mixtures; Detection; Development; Diagnosis; Diagnostic; Disease; Early Intervention; Enzymes; Epitopes; Gold; Heart; Heart Diseases; Heart failure; Injury; Intervention; Ligand Binding; Ligands; Link; Liquid Chromatography; Mass Spectrum Analysis; Medical; Methods; Modification; Morbidity - disease rate; Myocardial Infarction; Nanotechnology; Necrosis; Peptides; Phase; Protein Binding; Proteins; Proteomics; Quality of life; Reporting; Reproducibility; Research; Resolution; Scheme; Silicon Dioxide; Staging; Surface; Tissue Extracts; Tissue Sample; Tissues; Troponin; Troponin I; United States; Variant; Water; Western Blotting; Woman; accurate diagnosis; base; cost; design; experience; functional loss; global health; heart cell; improved; interdisciplinary approach; iron oxide; magnetite ferrosoferric oxide; men; mortality; nanomaterials; nanoparticle; novel strategies; polypeptide; protein complex; public health relevance; success

 DESCRIPTION (provided by applicant): Heart failure is the leading cause of morbidity and mortality in the United States and is becoming a global health problem. Accurate diagnosis of the disease at the early stage will allow implementation of early intervention strategies that will not only reduce medical costs, but also enable successful treatment of the disease and improve the quality of life. Cardiac troponin I (cTnI), the inhibitory subunit of troponin complex (cTnI-T-), is the gold standard biomarker in the detection of cardiac injuries. However, there are significant challenges in creating a standard and reliable assay using cTnI. One of these challenges is that the commercially available assays for cTnI detection typically use antibodies to target various epitopes of cTnI with varied quality, yielding inconsistent results. Moreover, cTnI typically exist in low abundance but in a myriad forms (phosphorylated, oxidized, degraded, in I-T-C or I-C complex as well as free forms) once released into the blood, which presents a significant challenge for a single antibody-based approach. Herein, I propose to develop a comprehensive cardiac troponin assay enabled by nanotechnology and top-down proteomics. Specifically, I will develop nanoparticles (NPs) functionalized with a range of cTnI affinity ligands to target various epitopes of cTnI to enable the enrichment of the low abundant cTnI proteins from complex blood samples for more accurate detection, effective separation, and subsequent quantitative analysis by high-resolution top-down mass spectrometry. These "artificial antibodies" will eliminate the batch-to-batch variability of the antibodies currently used, allowing for a more reliable assay. Specifically, I will first construct multifunctional superparamagnetic NPs with cTnI binding moieties displayed on the surface. The basic design will consist of iron oxide NPs coated with a silica shell, a fluorescent marker (for detection), cleavable moiety (detachment of cTnI for analysis), and the affinity ligand (for binding with cTnI). After the synthesis of the nanomaterial I will evaluate and demonstrate these functionalized NPs for the enrichment and detection of cTnI from heart tissue, and then blood samples. To rapidly detect the cTnI and related forms, we will develop both Western blot and enzyme-linked immunosorbant assay (ELISA) with these NP-based "artificial antibodies" that contain fluorescent moieties. The captured intact cTnI proteins will be further separated by liquid chromatography and analyzed by high resolution mass spectrometry-based top-down proteomics. The success of the proposed research will help in the development of a more reliable and accurate assay for the detection of cTnI that would result in a better diagnostic of heart disease.

 描述(由申请人提供)：心力衰竭是美国发病率和死亡率的主要原因，并正在成为一个全球健康问题。在疾病的早期阶段进行准确的诊断将有助于实施早期干预策略， 不仅降低了医疗成本，而且使疾病得到了成功的治疗，提高了生活质量。心肌肌钙蛋白I(CTnI)是肌钙蛋白复合体(cTnI-T-)的抑制亚单位，是检测心脏损伤的金标准生物标志物。然而，有重要的 使用cTnI创建标准和可靠的检测面临的挑战。这些挑战之一是，商业上可用的cTnI检测方法通常使用抗体来靶向质量不同的cTnI的各种表位，结果不一致。此外，cTnI通常以低丰度存在，但一旦释放到血液中就以多种形式(磷酸化、氧化、降解、I-T-C或I-C复合体以及游离形式)存在，这对基于单一抗体的方法构成了重大挑战。在此，我建议开发一种利用纳米技术和自上而下的蛋白质组学来实现的全面的心肌肌钙蛋白检测。具体地说，我将开发带有一系列cTnI亲和配体的纳米颗粒(NPs)，以靶向cTnI的各种表位，从而能够从复杂的血液样本中浓缩低丰度的cTnI蛋白，以便更准确地检测、有效分离，并随后通过高分辨率自上而下的质谱学进行定量分析。这些“人造抗体”将消除目前使用的抗体在批次之间的可变性，从而实现更可靠的检测。具体地说，我将首先构建多功能超顺磁性纳米粒子，并在表面显示cTnI结合部分。基本设计将包括包覆二氧化硅外壳的氧化铁纳米颗粒、荧光标记(用于检测)、可切割部分(用于分析cTnI的分离)和亲和配体(用于与cTnI结合)。在纳米材料合成后，我将评估和展示这些功能化的NPs，用于从心脏组织和血液样本中浓缩和检测cTnI。为了快速检测cTnI和相关形式，我们将用这些含有荧光部分的NP基人工抗体建立Western印迹和酶联免疫吸附试验(ELISA)。捕获的完整cTnI蛋白将进一步用液相色谱分离，并用基于高分辨率质谱学的自上而下的蛋白质组学进行分析。拟议研究的成功将有助于开发一种更可靠、更准确的cTnI检测方法，从而更好地诊断心脏病。