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.

质谱法已用于确定修饰的程度和生物分子修饰的特定位点。基于MS的方法具有许多优势，包括通常不进行放射性标记的快速分析。已经使用质谱法研究了多种蛋白质的MS分析。通过正离子MALDI质谱法和LC结合电喷雾质谱法对产物和消化进行了分析。此外，我们目前正在分析使用多种蛋白质的交联BS3的使用。 1。抗体糖基化表征。针对骨髓氧化酶（MPO）和蛋白酶3（PR3）的抗中性嗜性胞质自身抗体（ANCA）在ANCA肾小球肾炎和血管炎（AAV）中被认为是致病性的。 ANCA主要是IgG同种型。 IgG分子在其每个FC CH2结构域中都包含N-聚糖。此外，15-25％的血清IgG在FAB变量域中包含糖。据报道，血清IgG糖型概况的修饰，尤其是在FC区域，已被报道为包括ANCA疾病在内的几种炎症自身免疫性疾病的发病机理的一个因素。但是，IgG FC和Fab糖基化在ANCA致病性中的各自作用在很大程度上没有探索。我们在IgG FC部分上的疾病活性变化方面显示了MPO-和PR3-ANCA疾病患者之间的显着差异。与非运动反应性IgG相比，抗MPO特异性IgG上Fab糖基化位点的患病率显着增加。 2。组蛋白蛋白。当我们淘汰H1基因时，我们一直在与弓箭手实验室合作，以研究H1在控制基因表达和蛋白质水平中的作用。组蛋白H1.4蛋白的基因敲除细胞系（由MS先前确定为磷酸化）与“野生型”细胞系进行了比较。获取数据并正在分析，以确定如果H1.4同工型被淘汰，其他组蛋白水平是否改变和/或其PTMS会改变。 3。蛋白质交联。正在进行多个项目，以通过质谱和化学交联的结合来表征蛋白质复合物。这些实验大多数是使用BS3作为交联试剂进行的，其次是胰蛋白酶消化，并在Q-极性加质量光谱仪上进行的Nanolc-Esi-MS。尽管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综合体的表征。新发现的内核核酸酶（RNase）LAS1和多核苷酸激酶（PNK）GRC3聚集到多烯酶复合物中。为了了解GRC3-LAS1复合物如何协调其双重酶，使用了多种结构方法，包括化学交叉链接和质谱以及冷冻电子显微镜。解决了多个结构，它们揭示了GRC3-LAS1复合物采用蝴蝶样建筑，该结构具有复合材料HEPN核酸酶的活性位点，该结构是离散的RNA激酶位点的两侧。 4。VRK1的磷酸化。疫苗相关的激酶1（VRK1）是一种SER-THR激酶，可调节许多蛋白质。我们发现，在体外激酶测定中，VRK1自动磷酸化在Ser376和Thr386处呈磷酸化。发现在细胞中，THR386在低葡萄糖（40 mg/dL）但不高葡萄糖（140 mg/dl）培养基中被磷酸化。这种TH386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。在低葡萄糖培养基中，VRK1的敲低可减轻C-Jun和p53的磷酸化，而VRK1 T386D的过表达（但不表达VRK1 WT或VRK1 T386A突变体）诱导p53和C-Jun的磷酸化p53和C-Jun在高葡萄糖中的磷酸化。 5。花生蛋白质组学。与B. London和G. Mueller合作，我们以前曾使用质谱法研究了主要花生过敏原的年龄修饰。当前工作的目的是开发对潜在年龄生物标志物的蛋白质组学筛查，以了解热加工对花生的影响。