Proteomic Analysis for Biomarker Discovery in Autoimmune Disease

自身免疫性疾病生物标志物发现的蛋白质组学分析

• 批准号: 7968997
• 负责人: NICO TJANDRA
• 金额: $ 18.49万
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7968997
• 关键词: Affinity; Antibodies; Antigen-Antibody Complex; Antigens; Autoantibodies; Autoimmune Diseases; Binding; Biological Assay; Biological Markers; Cells; Chronic Lymphocytic Leukemia; Clinical; Collaborations; Complex; Cooperative Human Tissue Network; Dermatomyositis; Disease; Ensure; Event; Gel; Human; Immune; Immune system; Inflammatory; Ion Exchange; Mass Spectrum Analysis; Mediating; Molecular Weight; Myosin Heavy Chains; Myositis; National Institute of Neurological Disorders and Stroke; Patients; Plasma; Post-Translational Protein Processing; Postdoctoral Fellow; Precipitation; Proteins; Proteomics; Running; Sampling; Sodium Chloride; Solutions; Source; Spastic Paraplegia; Stress; Structure; Surface; System; Systemic Lupus Erythematosus; Tissues; base; disorder control; inflammatory marker; protein profiling; receptor; systemic autoimmune disease

A project beginning last year and continuing to date, was started by postdoctoral fellow Leticia Cano. It attempts to identify biomarkers in autoimmune disease using protein profiling in combination with mass spectrometry. Proteins isolated from tissue and immune complexes are used as a source of biomarkers. Collaborations have been established with several clinical groups involved in Sjogren's disease, systemic lupus erythematosus and inflammatory myositis (G. Illei (NICDR), F. Miller (NEIHS), E. Rushing (AFIP), Anil Chauhan (Progen Biologics), Craig Blackstone (NINDS), and Howard Jaffe (NINDS). We have also received OHSR approval to obtain human samples from the Cooperative Human Tissue Network (CHTN) and AFIP (Rushing). Tissues obtained from healthy controls and disease controls have been examined to ensure that we are not merely identifying markers of inflammation. New ways to separate and profile proteins prior to LC/MS/MS analysis are also being investigated, i.e., chromatofocusing, ion exchange, salt precipitations and affinity columns. As an example of the latter, we have isolated circulating immune complexes (CICs) from the plasma of patients with dermatomyositis (DM) and systemic lupus erythematosus (SLE) using a receptor-based affinity column from ProGen Biologics (Chauhan). We have found many proteins that were previously identified as candidate biomarkers for autoimmune diseases. The presence of these proteins in circulating immune complexes suggests that they are targeted by the immune system and that autoantibody assays for these proteins may be developed as biomarkers for systemic autoimmune diseases. We have begun using ELISAs to verify these findings and to show the utility of this approach in defining new biomarkers for immune-mediated diseases. A Beckman PF2D system for 2D-HPLC separation of intact proteins has been acquired and will be used for examining the spastic paraplegia samples (Blackstone). In a recent collaborations we identified a 225kDa marker belonging to myosin heavy chain IIA (MYHIIA) that reacts specifically with chronic lymphocytic leukemia antibody. The MYHIIA form structures on the surface of the HEp-2 cells which are undergoing stress. This suggests that chronic lymphocytic leukemia antibody may bind to surface markers that are the consequence of stress related cell events.

一个从去年开始并持续至今的项目是由博士后研究员莱蒂西亚卡诺启动的。它试图使用蛋白质谱结合质谱法来鉴定自身免疫性疾病中的生物标志物。从组织和免疫复合物中分离的蛋白质被用作生物标志物的来源。已经与涉及干燥病、系统性红斑狼疮和炎性肌炎的几个临床小组建立了合作关系（G。Illei（NICDR），F.米勒（NEIHS），E. Rushing（AFIP）、Anil Chauhan（Progen Biologics）、克雷格黑石（NINDS）和霍华德贾菲（NINDS）。我们还获得了OHSR批准，可以从合作人体组织网络（CHTN）和AFIP（Rushing）获取人体样本。 已经检查了从健康对照和疾病对照中获得的组织，以确保我们不仅仅是识别炎症标志物。 也正在研究在LC/MS/MS分析之前分离和分析蛋白质的新方法，即，色谱聚焦、离子交换、盐沉淀和亲和柱。作为后者的一个例子，我们使用来自ProGen Biologics（Chauhan）的基于受体的亲和柱从皮肌炎（DM）和系统性红斑狼疮（SLE）患者的血浆中分离出循环免疫复合物（CIC）。我们已经发现了许多以前被确定为自身免疫性疾病的候选生物标志物的蛋白质。这些蛋白质在循环免疫复合物中的存在表明它们被免疫系统靶向，并且针对这些蛋白质的自身抗体测定可被开发为系统性自身免疫疾病的生物标志物。 我们已经开始使用ELISA来验证这些发现，并显示这种方法在定义免疫介导疾病的新生物标志物方面的实用性。 已获得Beckman PF 2D系统，用于完整蛋白质的2D-HPLC分离，并将用于检查痉挛性截瘫样本（黑石）。 在最近的合作中，我们确定了一个属于肌球蛋白重链IIA（MYHIIA）的225 kDa标记物，该标记物与慢性淋巴细胞白血病抗体特异性反应。MYHIIA在经历应激的HEp-2细胞的表面上形成结构。这表明慢性淋巴细胞白血病抗体可能与应激相关细胞事件的结果表面标记物结合。