Proteomic Analysis for Biomarker Discovery in Autoimmune Disease

一个项目始于去年，至今仍在继续，由博士后莱蒂西亚·卡诺启动。它试图使用蛋白质图谱和质谱学相结合的方法来识别自身免疫性疾病的生物标记物。从组织和免疫复合体中分离出来的蛋白质被用作生物标记物的来源。已经与几个涉及干燥病、系统性红斑狼疮和炎症性肌炎的临床小组建立了合作关系(G.Illei(NICDR)、F.Miller(NEIHS)、E.Russing(AFIP)、Anil Chauhan(Progen Biologics)、Craig Blackstone(NINDS)和Howard Jaffe(NINDS)。我们还获得了OHSR的批准，可以从合作人体组织网络(CHTN)和AFIP(Rashing)获取人体样本。从健康对照和疾病对照中获得的组织已经进行了检查，以确保我们不仅仅是在识别炎症的标志。 在LC/MS/MS分析之前分离和分析蛋白质的新方法也在研究中，例如，色谱聚焦、离子交换、盐析和亲和柱法。作为后者的一个例子，我们使用Progen Biologics(Chauhan)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体(CIC)。我们已经发现了许多以前被确定为自身免疫性疾病候选生物标记物的蛋白质。循环免疫复合体中这些蛋白质的存在表明它们是免疫系统的靶标，这些蛋白质的自身抗体检测可能被开发为系统性自身免疫性疾病的生物标记物。我们已经开始使用ELISA来验证这些发现，并展示这种方法在定义免疫介导性疾病的新生物标记物方面的实用性。 Beckman PF2D系统已用于完整蛋白质的2D-HPLC分离，并将用于检测痉挛性截瘫样本(Blackstone)。 在最近的一次合作中，我们发现了一个225 kDa的标记，属于肌球蛋白重链IIA(MYHIIA)，它能与慢性淋巴细胞白血病抗体发生特异性反应。MYHIIA在承受压力的Hep-2细胞表面形成结构。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志物结合。

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.