Methods In Mass Spectrometry

质谱分析方法

• 批准号: 8342082
• 负责人: SANFORD P MARKEY
• 金额: $ 72.89万
• 依托单位: NATIONAL INSTITUTE OF MENTAL HEALTH
• 依托单位国家: 美国
• 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/8342082
• 关键词: Affect; Affinity; Ala-Trp-Arg-His-Pro-Gln-Phe-Gly-Gly; Algorithms; Amino Acids; Architecture; Aspartic Acid; Binding; Bioinformatics; Biological; Biological Assay; Carbohydrates; Cells; Chemicals; Chimeric Proteins; Cloning; Code; Complex; Computer software; Computers; DNA; Data; Data Set; Databases; Engineering; Environment; Enzymes; Escherichia coli; Genes; Genetic Transcription; Goals; Human; Individual; Investigation; Isotope Labeling; Isotopes; Knock-out; Label; Laboratories; Learning; Length; Link; Lipids; Liquid Chromatography; Liquid substance; Literature; Mass Spectrum Analysis; Measurement; Measures; Memory; Messenger RNA; Methods; Mining; Molecular; Monitor; Neurons; Organelles; Peptides; Performance; Positioning Attribute; Post-Translational Protein Processing; Process; Property; Protein Analysis; Proteins; Proteomics; Psychotropic Drugs; Reaction; Regulon; Relative (related person); Research Personnel; Research Project Grants; Resolution; Ribosomes; Running; Sampling; Screening procedure; Serum Albumin; Software Tools; Synapses; Synthetic Genes; System; Techniques; Temperature; Testing; Time; Translations; United States National Institutes of Health; Urine; Water; analytical tool; arginyllysine; base; computerized data processing; expression vector; improved; instrument; knockout gene; liquid chromatography mass spectrometry; mass analyzer; mass spectrometer; molecular mass; multiple reaction monitoring; open source; physical separation; protein structure; repository; stoichiometry; synaptic function; tool

Mass spectrometric methods for the analysis of protein structures are being improved, applied and tested in independent and collaborative research projects in proteomics. Projects in separation methods and data processing are in progress. In the past year, progress has been made on implementation of a computational environment for mass spectrometric proteomic data processing; isotope labeled fusion proteins as protein standards; quantification of identified and unidentified components in sets of complex liquid chromatography-high resolution mass spectrometry data; understanding post translational modification in bacterial ribosomes; and multi-institutional collaborative studies on standards in proteomics. The processing and mining of mass spectrometric proteomic data requires a system allowing investigators desktop computer access to terabytes of data; the ability to repeatedly probe their data with emerging software tools; and the ability to transfer this data to public archival repositories. A versatile system has been implemented in LNT and will be shared with other proteomics groups in NIH. We sought to determine if biosynthetic concatenated labeled peptides (concatemers) are equivalent to whole labeled proteins as internal standards for isotope dilution mass spectrometry using selected reaction monitoring on a triple quadrupole mass analyzer. Mass spectrometry provides a platform for these measurements by using multiple reaction monitoring to follow specific transitions of peptides as they fragment; however, internal standards are only accurate if they faithfully mimic proteolytic properties of full-length proteins. We selected signature peptides plus 12 amino acids (6 amino- and 6 carboxy-terminal) through mass spectrometric screening as well as the public databases and literature. Synthetic genes for the extended selected sequences are fused with affinity tags and expressed by cloning a synthetic gene into an expression vector and labeled using 13C and 15N arginine and lysine amino acids. A human serum albumin (HSA) concatemer was tested because native HSA is readily available as well as 15N-labeled full-length HSA as a laboratory standard. HSA concatemer concentration was measured with respect to a chemically synthesized strep tag 10-mer peptide using a standard curve. Time, temperature, and enzyme studies were optimized. Three of the five peptides in the HSA concatemer accurately mimic the tryptic properties of native HSA. We demonstrated that concatenated HSA peptides can be used as internal standards for the quantification of HSA in urine samples. Traditional immunoturbidimetric data provided comparable results. For the immunoturbidimetric assays, 50 μL of urine are consumed; for the selected reaction monitoring mass spectrometry method, less than 5 μL are consumed. New algorithms that serve as the basis for the bioinformatics tools used in the analysis of liquid chromatographic - tandem high resolution mass spectrometry data resulted in several open source software tools becoming viable alternatives to proprietary software. Unbiased protein analysis has been required to assess differences between control and treatment groups in several of our projects. The determination of relative expression levels of peptides in large data sets with biological replicates has remained challenging. The size and complexity of the data generated by LNT has consistently pushed the envelope of all the existing software. Replicate LC/MS/MS runs at three concentrations of the Universal Proteomics Standard (Sigma UPS1) were acquired in profile mode at a resolution of 60,000 on an LTQ-Orbitrap. Resultant spectra were analyzed using MZmine, XCMS, Mass++, and Progenesis LC-MS in both profile and centroid mode. Additional replicate LC/MS/MS runs at a set concentration of UPS1 at each of four increasing background concentrations of Human Serum Albumin were also acquired. Data was analyzed with respect to performance with large dynamic range, and ease/accuracy of LC/MS/MS alignment. Ideal-Q, a software tool which integrates a database search requirement with AMT based assignments was also used for the analysis. The integration of features results in overall stable ratios, although the statistical spread of the data is extremely variable. The use of Accurate Mass Tags to share database assignments across multiple replicates enables a more complete description of the data. Studies on the functional characterization of the post-translational modification (PTM) beta-methylthio-aspartic acid of the Escherichia coli at position 88 in protein S12 have been completed. There is a correlation between the presence of this PTM and the transcription of anaerobic genes belonging to the FNR regulon. Mass spectrometry analysis and affinity pull-down assays were used to identify two proteins RimO and YcaO that are specifically linked to interacting with and modifying S12. Gene knockouts for both proteins revealed a dramatic decrease in the modified form of S12 (the RimO knockout resulted in a complete absence of modified S12) and an overlapping decrease in transcription of genes belonging to the FNR regulon (determined by DNA expression microarray). Further investigation revealed that an absence of the PTM results in a substantial decrease in the abundance of the transcriptional factor FNR required for these genes to be expressed. It therefore seems likely that the S12 PTM affects the mRNA specific translation of FNR although the molecular details are unknown.

在蛋白质组学的独立和协作研究项目中，正在改善，应用和测试蛋白质结构分析的质谱方法。分离方法和数据处理中的项目正在进行中。在过去的一年中，在实施质谱蛋白质组学数据处理的计算环境方面取得了进展。同位素将融合蛋白标记为蛋白质标准品；在一组复杂的液相色谱高分辨率质谱数据中量化已识别和未识别的成分；了解细菌核糖体的翻译后修饰；以及有关蛋白质组学标准的多机构合作研究。 质谱蛋白质组学数据的处理和采矿需要一个允许调查人员桌面计算机访问数据的系统的系统；使用新兴软件工具反复探究数据的能力；以及将这些数据传输到公共档案存储库的能力。 LNT已实施了多功能系统，并将与NIH的其他蛋白质组学组共享。 我们试图确定生物合成串联标记的肽（辅导者）是否等同于整个标记的蛋白质作为同位素稀释质谱法的内部标准标准，该标准是使用三倍四倍体质量分析仪上的选定反应监测。 质谱法通过使用多个反应监测来遵循肽碎片的特定过渡，为这些测量提供了一个平台。但是，只有在忠实地模仿全长蛋白的蛋白水解特性的情况下，内部标准标准才是准确的。 我们通过质谱筛选以及公共数据库和文献选择了签名肽以及12种氨基酸（6个氨基和6个羧基末端）。 扩展选定序列的合成基因与亲和标签融合，并通过将合成基因克隆到表达载体中表达，并使用13C和15N精氨酸和赖氨酸氨基酸标记。 测试了人血清白蛋白（HSA）助剂，因为本机HSA容易获得以及15N标记的全长HSA作为实验室标准。 使用标准曲线测量了相对于化学合成的链球菌标签10-Mer肽的HSA con缩浓度。 优化了时间，温度和酶研究。 HSA con夫的五个肽中的三个准确地模仿天然HSA的胰蛋白酶特性。 我们证明，串联的HSA肽可以用作尿液样品中HSA的内部标准。 传统的免疫尿素数据提供了可比的结果。 对于免疫甲型法测定，消耗了50μl的尿液；对于选定的反应监测质谱法，消耗少于5μL。 作为液态色谱分析的生物信息学工具的基础的新算法 - 串联高分辨率质谱数据导致几种开源软件工具成为专有软件的可行替代方案。需要公正的蛋白质分析来评估我们的几个项目中对照组和治疗组之间的差异。 在具有生物学重复的大数据集中确定肽的相对表达水平仍然具有挑战性。 LNT生成的数据的大小和复杂性一直推动所有现有软件的信封。以三个浓度的通用蛋白质组学标准（Sigma UPS1）在轮廓模式下以60,000的分辨率在LTQ-OrbitRap上以60,000的分辨率获取了复制的LC/MS/MS运行。使用Mzmine，XCM，质量++和祖发LC-MS分析所得的光谱。还获得了额外的重复LC/MS/MS在四个增加的背景浓度的人血清白蛋白的浓度下以UPS1的设定浓度运行。分析了具有较大动态范围的性能的数据，以及LC/MS/MS比对的易于/准确性。 Ideas-Q是一种将数据库搜索要求与基于AMT的作业集成的软件工具，也用于分析。 尽管数据的统计扩展非常可变，但功能的集成会导致总体稳定比率。使用准确的质量标签在多个重复方面共享数据库分配，可以更完整地描述数据。 关于蛋白质S12中第88个位置的大肠杆菌的翻译后修饰（PTM）β-甲基硫硫代基本酸的功能表征的研究已完成。 该PTM的存在与属于FNR调节的厌氧基因的转录之间存在相关性。 质谱分析和亲和力下拉测定法用于识别两种蛋白质RIMO和YCAO，这些蛋白与与S12相互作用并修改S12特别相关。 两种蛋白质的基因敲除均显示S12的修饰形式急剧下降（RIMO敲除导致完全没有修饰的S12），并且属于FNR调节的基因的转录重叠下降（由DNA表达微阵列确定）。 进一步的研究表明，缺乏PTM导致这些基因表达所需的转录因子FNR的丰度大大降低。 因此，尽管分子细节未知，但S12 PTM似乎可能影响FNR的mRNA特异性翻译。