Mass Spectrometry Characterization

质谱表征

基本信息

批准号：
10012714
负责人：
Leesa Deterding
金额：
$ 65.83万
依托单位：
NATIONAL INSTITUTE OF ENVIRONMENTAL HEALTH SCIENCES
依托单位国家：
美国
项目类别：
财政年份：
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/10012714
关键词：
Acetylation Active Sites Adopted Affect Allergens Amino Acids Antibodies Architecture Attenuated Autoantibodies Autoimmune Diseases Binding Biological Assay Biological Markers Butterflies Cell Line Cells Cellular biology Chemicals Collaborations Complex Cross-Linking Reagents Crosslinker Cryoelectron Microscopy Data Digestion Disease Endoribonucleases Enzymes Evaluation Gene Expression Gene Proteins Genes Glomerulonephritis Glucose Goals Histones Immunoglobulin G In Vitro Inflammatory Ions JUN gene Knock-out Laboratories Life Cycle Stages Lipids London Mass Spectrum Analysis Mediating Methylation Modification Molecular Molecular Conformation Molecular Structure Multienzyme Complexes Normal Cell Pathogenesis Pathogenicity Patients Peptides Peroxidases Phosphorylation Phosphotransferases Polynucleotide 5&apos -Hydroxyl-Kinase Polysaccharides Post-Translational Protein Processing Prevalence Prevention Protein Isoforms Proteinase 3 Proteins Proteome Proteomics RNA Radiolabeled Reporting Ribonucleases Role Safety Sampling Serum Side Site Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization Structure System TP53 gene Trypsin Ubiquitination Vaccinia Vasculitis Work base cell type crosslink experimental study functional group glycosylation insight knock-down mass spectrometer mutant neutrophil nuclease overexpression protein complex protein crosslink screening

项目摘要

Mass spectrometry has been used to determine the extent of modification and the specific sites of modification on biomolecules. MS-based approaches have many advantages, including generally rapid analyses without radiolabeling. The MS analysis of a variety of proteins has been investigated using mass spectrometry. Products and digests have been analyzed by both positive and negative ion MALDI mass spectrometry and LC in combination with electrospray mass spectrometry. In addition, we are currently analyzing the use of the crosslinker BS3 with a variety of proteins. 1. Antibody Glycosylation Characterization. Anti-neutrophil cytoplasmic autoantibodies (ANCA) directed against myeloperoxidase (MPO) and proteinase 3 (PR3) are considered pathogenic in ANCA glomerulonephritis and vasculitis (AAV). ANCAs are predominantly of the IgG isotype. IgG molecules contain an N-glycan on each of their Fc CH2 domains. In addition, 15-25%of serum IgG contains glycans within the Fab variable domain. Modification of the serum IgG glycoform profile, particularly in the Fc region, has been reported as a factor in the pathogenesis in several inflammatory autoimmune diseases including ANCA disease. However, the respective role of IgG Fc and Fab glycosylation in ANCA pathogenicity is largely unexplored. We showed significant differences between patients with MPO- and PR3-ANCA diseases with respect to changes with disease activity in the amount and type of glycans on the IgG Fc portion. The prevalence of Fab glycosylation sites on anti-MPO specific IgG is significantly increased compared to non-autoreactive IgGs. 2. Histone Proteins. We have been collaborating with the Archer laboratory in an effort to examine the role of H1 in controlling gene expression and protein levels when we knock out the H1 gene. Knockout cell lines of the histone protein H1.4 protein (as it was determined by MS previously that it was phosphorylated) was compared to a "wild-type" cell line. Data were acquired and are being analyzed to determine if other histone levels change and/or their PTMS change if the H1.4 isoform is knocked out. 3. Protein Crosslinking. Multiple projects are underway to characterize protein complexes by mass spectrometry in conjunction with chemical cross-linking. Most of these experiments have been conducted using BS3 as the cross-linking reagent followed by trypsin digestion and nanoLC-ESI-MS performed on a Q-Exactive Plus mass spectrometer. While there have been successful analyses on multiple projects, the most mature of these projects has been the characterization of the Grc3-Las1 complex. The newly discovered endoribonuclease (RNase) Las1 and the poly-nucleotide kinase (PNK) Grc3 assemble into the multienzyme complex. In an effort to understand how the Grc3-Las1 complex coordinates its dual enzymes, multiple structural approaches were used including chemical cross-linking and mass spectrometry as well as cryo-electron microscopy. The multiple structures were solved, and they reveal that the Grc3-Las1 complex adopts a butterfly-like architecture harboring a composite HEPN nuclease active site flanked by discrete RNA kinase sites. 4. Phosphorylation of VRK1. Vaccinia-related kinase 1 (VRK1) is a Ser-Thr kinase and regulates numerous proteins. We found that VRK1 auto-phosphorylates at Ser376 and Thr386 in in vitro kinase assays. It was found that in cells, that Thr386 is phosphorylated in the low glucose (40 mg/dL) but not high glucose (140 mg/dL) media. This Thr386 phosphorylation correlates with an induced phosphorylation of p53 (Thr18) and C-Jun (Ser63) in the low glucose medium. Knock down of VRK1 attenuates phosphorylation of both C-Jun and p53 in the low glucose medium, while over-expression of VRK1 T386D (but not VRK1 WT or VRK1 T386A mutant) induces phosphorylation of p53 and C-Jun in the high glucose medium. 5. Peanut Proteomics. In collaboration with B. London and G. Mueller, we have previously investigated the AGE modifications of the major peanut allergens using mass spectrometry. The goal of the current work is to develop a proteomic screening of potential AGE biomarkers for understanding the effects of thermal processing on peanuts.

质谱法已用于确定生物分子的修饰程度和修饰的具体位点。基于 MS 的方法有许多优点，包括无需放射性标记即可快速分析。已使用质谱法研究了多种蛋白质的 MS 分析。产物和消化物已通过正离子和负离子 MALDI 质谱以及 LC 与电喷雾质谱相结合进行了分析。此外，我们目前正在分析交联剂 BS3 与多种蛋白质的使用。 1. 抗体糖基化表征。针对髓过氧化物酶 (MPO) 和蛋白酶 3 (PR3) 的抗中性粒细胞胞质自身抗体 (ANCA) 被认为是 ANCA 肾小球肾炎和血管炎 (AAV) 的致病性。 ANCA 主要是 IgG 同型。 IgG 分子的每个 Fc CH2 结构域上都含有一个 N-聚糖。此外，15-25% 的血清 IgG 在 Fab 可变域内含有聚糖。据报道，血清 IgG 糖型谱的改变，特别是 Fc 区的改变，是包括 ANCA 疾病在内的多种炎症性自身免疫性疾病发病机制的一个因素。然而，IgG Fc 和 Fab 糖基化在 ANCA 致病性中各自的作用很大程度上尚未被探索。我们发现 MPO- 和 PR3-ANCA 疾病患者之间 IgG Fc 部分聚糖数量和类型随疾病活动而变化的显着差异。与非自身反应性 IgG 相比，抗 MPO 特异性 IgG 上 Fab 糖基化位点的普遍性显着增加。 2.组蛋白。我们一直在与Archer实验室合作，努力研究当我们敲除H1基因时，H1在控制基因表达和蛋白质水平方面的作用。将组蛋白 H1.4 蛋白（之前通过 MS 确定其被磷酸化）的敲除细胞系与“野生型”细胞系进行比较。获取数据并进行分析，以确定如果 H1.4 同工型被敲除，其他组蛋白水平是否会发生变化和/或它们的 PTMS 会发生变化。 3.蛋白质交联。多个项目正在进行中，通过质谱法结合化学交联来表征蛋白质复合物。大多数这些实验都是使用 BS3 作为交联试剂进行的，然后进行胰蛋白酶消化，并在 Q-Exactive Plus 质谱仪上进行 nanoLC-ESI-MS。虽然对多个项目进行了成功的分析，但这些项目中最成熟的是 Grc3-Las1 复合体的表征。新发现的核糖核酸内切酶 (RNase) Las1 和多核苷酸激酶 (PNK) Grc3 组装成多酶复合物。为了了解 Grc3-Las1 复合物如何协调其双酶，使用了多种结构方法，包括化学交联、质谱以及冷冻电子显微镜。多重结构得到解决，揭示了 Grc3-Las1 复合物采用蝴蝶状结构，包含复合 HEPN 核酸酶活性位点，两侧是离散的 RNA 激酶位点。 4. VRK1 的磷酸化。牛痘相关激酶 1 (VRK1) 是一种 Ser-Thr 激酶，可调节多种蛋白质。我们在体外激酶测定中发现 VRK1 在 Ser376 和 Thr386 处自动磷酸化。研究发现，在细胞中，Thr386 在低葡萄糖 (40 mg/dL) 培养基中被磷酸化，但在高葡萄糖 (140 mg/dL) 培养基中则不会被磷酸化。 Thr386 磷酸化与低葡萄糖培养基中 p53 (Thr18) 和 C-Jun (Ser63) 的诱导磷酸化相关。 VRK1 的敲低会减弱低葡萄糖培养基中 C-Jun 和 p53 的磷酸化，而 VRK1 T386D（但不是 VRK1 WT 或 VRK1 T386A 突变体）的过表达会诱导高葡萄糖培养基中 p53 和 C-Jun 的磷酸化。 5.花生蛋白质组学。我们之前与 B. London 和 G. Mueller 合作，利用质谱法研究了主要花生过敏原的 AGE 修饰。当前工作的目标是开发潜在 AGE 生物标志物的蛋白质组筛选，以了解热处理对花生的影响。