Protein Sequencing by Tandem Mass Spectrometry

通过串联质谱法进行蛋白质测序

• 批准号: 8186745
• 负责人: DONALD F HUNT
• 金额: $ 70.13万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 1987
• 资助国家: 美国
• 起止时间: 1987-01-12 至 2015-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8186745
• 关键词: Acidic Amino Acids; Affinity Chromatography; Algorithms; Alkynes; Amines; Amino Acid Motifs; Amino Acid Sequence; Amino Acids; Antibodies; Binding; Cell Nucleus; Cell division; Cells; Centromere; Charge; Chemicals; Chemistry; Chromatin; Chromosome Segregation; Complex; Computer software; Cytoplasmic Granules; DNA; DNA Binding; Diabetes Mellitus; Digestion; Disease; Dissociation; Educational process of instructing; Electron Transport; Embryo; Enzymes; Epigenetic Process; Event; Genetic Transcription; Genomic Instability; Grant; Guanine Nucleotide Exchange Factors; Histone Code; Histone H3; Histones; Human; Hydrogen; Label; Location; Malignant Neoplasms; Mass Spectrum Analysis; Mental disorders; Metals; Methods; Methylation; Methyltransferase; Mitochondria; Mitochondrial Proteins; Modification; Mus; N-Acetylglucosamine kinase; N-terminal; Nuclear; Nuclear Proteins; Nucleosomes; Nucleotides; Nutrient; Pattern; Peptide Sequence Determination; Peptides; Phosphorylation; Phosphotyrosine; Polyploidy; Post-Translational Protein Processing; Prevention; Protein Fragment; Proteins; Protocols documentation; RNA-Binding Proteins; Reading; Research; Research Project Grants; Research Proposals; Sampling; Sequence Analysis; Signal Transduction; Site; Stress; Targeted Research; Techniques; Technology; Time; Undifferentiated; Variant; Work; Writing; centromere protein A; embryonic stem cell; improved; interest; repaired; research study; response; tandem mass spectrometry; titanium dioxide; zygote

DESCRIPTION (provided by applicant): The overall focus of this research project is to develop methods for characterizing protein post-translational modifications, particularly those that exist in combination on the same protein molecule and together regulate its activity. Examples include the hundreds, if not thousands, of different combinations of methyl, acetyl and phoshorylation marks that exist on histones (the "histone code"), and the crosstalk between phosphorylation and O-GlcNAcylation that occurs on many other proteins. Because only a small fraction of particular protein molecules will be post-translationally modified at any given time, protocols are required that include; (a) enrichment of the sample for the posttranslational modification of interest; (b) enzymatic digestion that converts the modified protein to the largest possible fragments that still cover the complete sequence of the protein; and (c) mass spectrometry technology that facilitates sequence analysis of fragments that contain 60-100 residues (or more) at a time and is able to assign the location of the most labile of posttranslational modifications to a single, amino-acid residue. Electron transfer dissociation (ETD) mass spectrometry, a technique developed in the previous grant period, is ideally suited for this purpose. Since ETD works best on multiply charged protein fragments, we will evaluate several enzymatic and chemical methods for increasing the charge state of peptides and proteins by converting neutral or acidic, amino-acid residues to amines that can then be protonated and function as hydrogen donors. Also proposed are several new ways of tagging OGlcNAcylated proteins so that they can be enriched from complex sample mixtures by immobilized metal affinity chromatography. In addition to the above work, we will conduct experiments to define the posttranslational modifications that exist on the histone, H3 variant, CENP-A, that is located in nucleosomes at the centromere and also continue our efforts to characterize post-translational modifications on histone and histone binding partners involved in establishing the epigenetic state (modified chromatin) in the fertilized egg. We will also characterize the substrate motif for a protein n-terminal methyltransferase, NRMT, identify human nuclear proteins that are trimethylated on the n-terminus, and identify and characterize the demethylase that removes this modification. We have already determined that RCC1, a nucleotide exchange factor that binds to histones and DNA, is methylated on its n-terminus. This modification is required for the prevention of genomic instability (polyploidy nuclei). Two other important proteins, retoblastoma B and CENP-A, are both trimethylated as well. We believe that several hundred DNA and RNA binding proteins are likely to be modified in the same way. Accordingly, we will conduct experiments to identify these proteins and determine the n-terminal motif required for trimethylation by NRMT. PUBLIC HEALTH RELEVANCE: Proposed here is research to develop mass spectrometry methods to identify combinations of posttranslational modifications on proteins that are involved in cell signaling. Targets for this research are proteins that regulate DNA transcription, silencing, and repair plus proper segregation of chromosomes during cell division. Proteins that regulate the cellular response to stress and nutrient availability, and those involved in establishing the embryonic epigenetic state, will also be targeted. Dysregulation of cell signaling is a hallmark of many disease states, including diabetes, cancer, and numerous mental disorders.

描述（由申请人提供）：该研究项目的总体重点是开发表征蛋白质后翻译后修饰的方法，尤其是那些在同一蛋白质分子上合并并共同调节其活性的方法。例子包括组蛋白上存在的甲基，乙酰基和哲学标记的数百种（即使不是数千个），以及在许多其他蛋白质上发生的磷酸化与O-Glcnacylation之间的串扰。因为在任何给定时间，只有一小部分特定蛋白质分子会在后翻译后修饰，因此需要进行协议。 （a）富集样品的转变后修饰； （b）将修饰的蛋白质转化为仍覆盖蛋白质完整序列的最大片段的酶消化； （c）质谱技术促进了一次含有60-100个残基（或更多）的片段的序列分析，并且能够为单个氨基酸残留物分配最不稳定的翻译后修饰的位置。电子转移解离（ETD）质谱是在上一个赠款期间开发的一种技术，非常适合此目的。由于ETD最适合多重带电的蛋白质片段，因此我们将通过将中性或酸性的，氨基酸残基转化为可以被质子化并充当氢供体的胺，评估几种酶和化学方法，以增加肽和蛋白质的电荷状态。还提出的是标记Oglcnacylated蛋白的几种新方法，以便可以通过固定的金属亲和色谱从复杂的样品混合物中富集它们。 In addition to the above work, we will conduct experiments to define the posttranslational modifications that exist on the histone, H3 variant, CENP-A, that is located in nucleosomes at the centromere and also continue our efforts to characterize post-translational modifications on histone and histone binding partners involved in establishing the epigenetic state (modified chromatin) in the fertilized egg.我们还将表征蛋白质N末端甲基转移酶NRMT的底物基序，鉴定在N末端对甲基化的人类核蛋白质，并鉴定和表征去除这种修饰的脱甲基酶。我们已经确定RCC1是与组蛋白和DNA结合的核苷酸交换因子，在其N末端被甲基化。这种修饰是预防基因组不稳定性（多倍体核）所必需的。另外两个重要的蛋白质B和CENP-A也是三甲基化的。我们认为，几百个DNA和RNA结合蛋白可能以相同的方式修饰。因此，我们将进行实验以鉴定这些蛋白质并确定NRMT三甲基化所需的N末端基序。 公共卫生相关性：此处提出的是开发质谱方法的研究，以鉴定翻译后修饰的组合对细胞信号传导所涉及的蛋白质。这项研究的靶标是调节DNA转录，沉默和修复以及细胞分裂过程中染色体的适当分离的蛋白质。调节细胞对胁迫和养分利用率的反应的蛋白质以及涉及建立胚胎表观遗传态的蛋白质也将被靶向。细胞信号传导失调是许多疾病状态的标志，包括糖尿病，癌症和许多精神疾病。