Mass Spectrometry Identification

质谱鉴定

基本信息

批准号：
10925013
负责人：
Jason Williams
金额：
$ 142.79万
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/10925013
关键词：
Acetylation Acetyltransferase Affect Albumins Amino Acids Arginine Autophagocytosis Base Excision Repairs Binding Bleomycin Cell Line Cells Cellular biology Chemicals Chimeric Proteins Collaborations Compensation Complex Coupled Cross-Linking Reagents Crosslinker Cryoelectron Microscopy DNA Repair Gene Data Deacetylase Deposition Development Digestion Disease Dissociation E1A-associated p300 protein EP300 gene Elements Enzymes Evaluation Event Fibroblasts Functional disorder Gel Gene Expression Gene Proteins Genes Glomerulonephritis Histone H1 Histones Human IGA Glomerulonephritis IgA1 Immunoglobulin A In Vitro Intramural Research Ions Kidney Diseases Knock-out Label Laboratories Life Cycle Stages Link Lung Lysine Malignant Neoplasms Mass Spectrum Analysis Mediating Methylation Minnesota Modification Molecular Molecular Structure Mus Nature Normal Cell Outcome Pathogenesis Patients Peptide Hydrolases Peptides Phosphorylation Plasma Proteins Platelet-Derived Growth Factor alpha Receptor Polymerase Polymers Post-Translational Protein Processing Preparation Prevention Principal Investigator Proteins Proteome Proteomics Publications RNA Splicing Radiolabeled Reducing Agents Renal Hypertension Research Research Project Grants Resolution Role Sampling Scientist Serine Services Side Signal Transduction Pathway Site Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization Structure Structure of glomerular mesangium Sulfur Support Groups System Techniques Threonine Transfer RNA Transferase Trypsin Tyrosine Phosphorylation Ubiquitination Universities Variant Western Blotting alpha 1-Antitrypsin alpha-1-microglobulin cell type crosslink disulfide bond experimental study glycosylation histone modification insight lipidomics mass spectrometer molecular mass osteosarcoma overexpression protein aggregation protein complex protein crosslink protein expression protein structure ran GTP-Binding Protein small molecule structural biology thioether

项目摘要

A variety of service and collaborative projects for mass spectrometry analyses have been or are being carried out within the Mass Spectrometry Research and Support Group with approximately 3000 samples analyzed from 39 scientists representing 26 principal investigators or core heads from 7 laboratory branches and the DTT. One large effort is in support of the protein expression function of the Structural Biology Core Laboratory and Dr. Bob Petrovich. The role of the MSRSG is to confirm gene expression at the protein level prior to the Structural Biology Core Laboratory handing materials over to their users. We have also put effort into the development of lipidomics. This project involves the identification/characterization as well as the quantitation of compounds; thus, this project is included in both the identification/characterization project and the quantitation project. In addition, 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. Additionally, the use of the crosslinker BS3 with a variety of proteins has been utilized. 1. Proteomic analysis of IgA-protein aggregates. We are currently involved in a collaborative effort between the Division of Renal Diseases and Hypertension from the University of Minnesota at Minneapolis. IgA nephropathy is the most common form of glomerulonephritis worldwide. The hallmark of the disease is deposition of IgA1 in the glomerular mesangium. These deposited IgA1 are mainly polymeric in nature and include heteromeric complexes of IgA covalently bound to other plasma proteins. Identification of the key constituents of these protein-protein complexes may lead to better understanding of the pathophysiology of this disease. Approximately 10% of IgA1 in archival samples from IgAN patients and controls were found as high molecular mass complexes. Immunoblotting demonstrated 1:1 complex between IgA and albumin, alpha-1-antitrypsin, or alpha-1-microglobulin. Some complexes dissociated completely to free proteins and IgA1 when reducing agents were added, suggesting that these proteins were covalently linked through disulfide bonds (SS bonds). We were able to identify several inter-chain SS between IgA1 and other plasma proteins. A nonreducible thioether bridge, resulting of elimination of one of the sulfur atoms from the disulfide bond, was also identified in one of the heteromeric protein complexes. 2. Histone Proteins. In collaboration 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 in human osteosarcoma cells. Knockout cell lines of the histone protein H1.4 protein (previously determined by MS to be phosphorylated) was compared to a "wild type" cell line. Data were acquired and showed 8 linker histone H1 proteins were confidently identified and quantified in the protein digests of the 3 types of samples. Significant sequence coverage (38-77%) was obtained for all linker histones identified. Label-free quantification using a MS1-based quantification approach confirms initial observations that the cells try to compensate for the loss of H1.4 by upregulating the other histone variants. 17 histone marks were confidently identified, consisting of 6 types of histone modifications: arginine methylation, lysine methylation, lysine acetylation, serine phosphorylation, threonine phosphorylation, and tyrosine phosphorylation. Eleven PTM-containing peptides were confidently quantified across all types of samples. Of these, 9 PTMs were up-regulated and 2 PTMs were down-regulated in the knockout lines relative to the parental wild-type cell line. As an outcome of these efforts, this collaboration has resulted in a publication. 3. Protein Crosslinking. Multiple projects have been analyzed to characterize protein complexes by mass spectrometry in conjunction with chemical cross-linking. 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, we recently identified chemical crosslinks in the TSEN-CLP1 complex. In efforts to understand the role of the TSEN and tRNA splicing, multiple approaches were used including chemical cross-linking and mass spectrometry as well as cryo-electron microscopy. In addition, in collaboration with the P. Blackshear group, we have analyzed the chemical crosslinks in Gaboon TTP and the CNOT1 proteins.Additionally, in collaboration with the R.S. Williams group, where we have analyzed the intramolecular crosslinks within BS3 treated Senataxin. Finally, in collaboration with the W. Copeland group, we have analyzed the intra- and inter-molecular BS3 crosslinks between polymerase gamma and lonP protease. 4. Characterization of PTMs. In 2023 we collaborated with Ms. Carol Trempus and Dr. Stavros Garantziotis to investigate the phosphorylation of proteins from pdgfr-alpha positive fibroblasts isolated from murine lungs after PBS or bleomycin treatment. We used a relative quantification approach to evaluate changes in phosphorylation of proteins in the samples with the hopes that changes in the phosphorylation events might shed light on what signal transduction pathways were altered by bleomycin treatment. We are also currently collaborating with Dr. Carlos Guardia in the analyses of glycosylation and other PTMs on Autophagy-Related Protein 9A and 9B. Moreover, in collaboration with Drs. Anirban Kar and Paul Doestch, we have been analyzing the in vitro acetylation of NTHL1. Acetylation on DNA repair proteins is a dynamic epigenic modification regulated by acetyltransferases and lysine deacetylases. Accumulating evidence has indicated that the aberrant acetylation of DNA repair proteins contributes to the pathogenesis of cancer. Hence, probing the acetylation states of DNA repair proteins might be of great help in better understanding pathogenesis of cancer. NTHL1 is a bifunctional glycosylase involved in base excision repair. We were asked to identify the acetylation sites on four truncated variants of NTHL1 that were overexpressed as fusions to the C-terminus of bacterial MBP. The MBP-NHTL1 fusion proteins were subjected to in vitro acetylation using p300 protein as acetyl transferase before running the protein on an SDS-PAGE gel. Proteins in the gel bands were then reduced, alkylated, and digested with trypsin. Tryptic digests were analyzed using UPLC coupled to an orbitrap high resolution mass spectrometer. Nine lysine acetylation sites were identified in the four types of NHTL-1 constructs whereas 26 sites were detected on MBP. There were no significant differences in lysine acetylation profiles between p300-treated variants and the corresponding non-acetylated control. Other smaller projects include characterization of stable domains, post translational modifications, and extent of chemical labeling for various intramural research groups.

质谱研究和支持小组已经或正在开展各种质谱分析服务和合作项目，对来自 39 名科学家（代表来自 7 个实验室分支机构和 DTT 的 26 位主要研究人员或核心负责人）的约 3000 个样品进行了分析。其中一项巨大的努力是支持结构生物学核心实验室和 Bob Petrovich 博士的蛋白质表达功能。 MSRSG 的作用是在结构生物学核心实验室将材料交给用户之前在蛋白质水平上确认基因表达。我们还致力于脂质组学的发展。该项目涉及化合物的鉴定/表征以及定量；因此，该项目既包含在鉴定/表征项目中，也包含在定量项目中。此外，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，还使用了交联剂 BS3 与多种蛋白质。 1. IgA-蛋白质聚集体的蛋白质组学分析。我们目前正在参与明尼苏达大学明尼阿波利斯分校肾脏疾病和高血压科之间的合作。 IgA 肾病是全世界最常见的肾小球肾炎形式。该疾病的标志是 IgA1 在肾小球系膜中沉积。这些沉积的 IgA1 本质上主要是聚合体，包括与其他血浆蛋白共价结合的 IgA 异聚复合物。鉴定这些蛋白质-蛋白质复合物的关键成分可能有助于更好地了解这种疾病的病理生理学。在 IgAN 患者和对照的档案样本中，大约 10% 的 IgA1 被发现为高分子复合物。免疫印迹证明 IgA 与白蛋白、α-1-抗胰蛋白酶或 α-1-微球蛋白之间存在 1:1 复合物。当添加还原剂时，一些复合物完全解离为游离蛋白质和 IgA1，这表明这些蛋白质通过二硫键（SS 键）共价连接。我们能够识别 IgA1 和其他血浆蛋白之间的几种链间 SS。在一种异聚蛋白复合物中还鉴定出了由于二硫键中的一个硫原子被消除而形成的不可还原的硫醚桥。 2.组蛋白。当我们敲除人类骨肉瘤细胞中的 H1 基因时，与 Archer 实验室合作，研究 H1 在控制基因表达和蛋白质水平中的作用。将组蛋白 H1.4 蛋白（之前通过 MS 确定为磷酸化）的敲除细胞系与“野生型”细胞系进行比较。获取的数据显示，在 3 种类型的样品的蛋白质消化物中，8 个接头组蛋白 H1 蛋白得到了可靠的鉴定和定量。所有已鉴定的接头组蛋白均获得了显着的序列覆盖率 (38-77%)。使用基于 MS1 的定量方法进行的无标记定量证实了最初的观察结果，即细胞试图通过上调其他组蛋白变体来补偿 H1.4 的损失。可靠地鉴定出 17 个组蛋白标记，包括 6 种组蛋白修饰：精氨酸甲基化、赖氨酸甲基化、赖氨酸乙酰化、丝氨酸磷酸化、苏氨酸磷酸化和酪氨酸磷酸化。在所有类型的样品中，11 种含有 PTM 的肽得到了可靠的定量。其中，相对于亲代野生型细胞系，敲除细胞系中 9 个 PTM 上调，2 个 PTM 下调。作为这些努力的成果，此次合作出版了一份出版物。 3.蛋白质交联。已经分析了多个项目，通过质谱法结合化学交联来表征蛋白质复合物。这些实验使用 BS3 作为交联试剂，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，但我们最近在 TSEN-CLP1 复合物中发现了化学交联。为了了解 TSEN 和 tRNA 剪接的作用，使用了多种方法，包括化学交联、质谱以及冷冻电子显微镜。此外，我们与 P. Blackshear 小组合作，分析了 Gaboon TTP 和 CNOT1 蛋白中的化学交联。 Williams 小组，我们分析了 BS3 处理的 Senataxin 内的分子内交联。最后，我们与 W. Copeland 小组合作，分析了聚合酶 γ 和 lonP 蛋白酶之间的分子内和分子间 BS3 交联。 4. PTM 的表征。 2023 年，我们与 Carol Trempus 女士和 Stavros Garantziotis 博士合作，研究了从小鼠肺部分离的 pdgfr-alpha 阳性成纤维细胞在 PBS 或博莱霉素处理后的蛋白质磷酸化情况。我们使用相对定量方法来评估样品中蛋白质磷酸化的变化，希望磷酸化事件的变化能够揭示博莱霉素治疗改变了哪些信号转导途径。我们目前还与 Carlos Guardia 博士合作分析自噬相关蛋白 9A 和 9B 的糖基化和其他 PTM。此外，与博士合作。 Anirban Kar 和 PaulDoestch，我们一直在分析 NTHL1 的体外乙酰化。 DNA 修复蛋白的乙酰化是一种受乙酰转移酶和赖氨酸脱乙酰酶调节的动态表观修饰。越来越多的证据表明 DNA 修复蛋白的异常乙酰化有助于癌症的发病机制。因此，探测DNA修复蛋白的乙酰化状态可能对更好地了解癌症的发病机制有很大帮助。 NTHL1 是一种参与碱基切除修复的双功能糖基化酶。我们被要求鉴定 NTHL1 的四个截短变体上的乙酰化位点，这些变体作为与细菌 MBP C 末端的融合体而过度表达。使用 p300 蛋白作为乙酰基转移酶对 MBP-NHTL1 融合蛋白进行体外乙酰化，然后在 SDS-PAGE 凝胶上运行该蛋白。然后将凝胶条带中的蛋白质还原、烷基化并用胰蛋白酶消化。使用与轨道阱高分辨率质谱仪联用的 UPLC 分析胰蛋白酶消化物。在四种类型的 NHTL-1 构建体中鉴定出 9 个赖氨酸乙酰化位点，而在 MBP 上检测到 26 个位点。 p300 处理的变体和相应的非乙酰化对照之间的赖氨酸乙酰化谱没有显着差异。其他较小的项目包括稳定结构域的表征、翻译后修饰以及各个校内研究小组的化学标记范围。