Methods In Mass Spectrometry

在蛋白质组学的独立和合作研究项目中，用于分析蛋白质结构的质谱方法正在得到改进、应用和测试。分离方法、衍生化化学和数据处理方面的项目正在进行中。通过LC-MS/MS进行的肽分析产生了大量的数据集，将肽和/或蛋白质鉴定信息整合为有意义的知识需要大量的劳动。软件生物信息学工具可以促进和加速高通量肽数据分析。为了实现这一目标，我们正在开发集成的工作流程软件，旨在最大限度地从LC-MS/MS数据中获得肽序列信息。该策略使用关系数据库体系结构集成了几种独立的数据搜索和数据挖掘技术。迭代搜索工具从数据库搜索算法中获取结果；光谱滤波和从头排序算法相结合，以最大限度地提高频谱分配。这些开源软件工具将为研究人员提供自动化的数据处理/分析环境。基于概率的搜索算法计算一个分数来表示观察到的光谱与从序列搜索库中计算出的光谱之间的匹配的统计显著性。多个搜索引擎可以增加肽序列搜索范围和/或识别冲突或模糊的谱分配。我们向开源软件包DBParser添加了新特性，以组合和验证来自OMSSA、Mascot和X！串联。DBParser生成组合的或比较的肽或蛋白质列表，供研究人员进行后续评估。蛋白质摘要基于简约原则，确定可以解释所有观察到的肽的最小蛋白质集。该程序生成排序和比较肽/蛋白质列表的html输出报告，可用于后续分析。基于cgi的图形用户界面允许使用web浏览器在网络上执行DBParser，以方便多个实验室使用它。DBParser已经过改进，以包含量化。一种测定巨蛋白复合物中蛋白质化学计量学的新技术已经被测试并正在改进。从线粒体或亚细胞器中分离的蛋白质复合物正在被探索，以确定最佳的分离和分析方法。美国国立卫生研究院设计和建造的一种新的荧光检测器正在用荧光肽衍生物进行定量和比较复杂混合物的测试。

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, derivatization chemistry, and data processing are in progress. Peptide analyses by LC-MS/MS generate large datasets, imposing labor-intensive efforts to consolidate peptide and/or protein identification information into meaningful knowledge. Software bioinformatics tools can facilitate and accelerate high-throughput peptide data analyses. Toward this goal, we are developing integrated workflow software designed to maximize the amount of peptide sequence information from LC-MS/MS data. This strategy integrates several independent data search and data mining technologies using a relational database architecture. An iterative search tool acquires results from database search algorithms; spectral filtering and de novo sequencing algorithms are integrated to maximize spectral assignment. These open source software tools will provide an automated data processing/analysis environment for researchers. Probability-based search algorithms calculate a score to indicate the statistical significance of a match between an observed spectrum and that calculated from a sequence search library. Multiple search engines may increase peptide sequence search coverage and/or identify conflicting or ambiguous spectral assignments. We added new features to an open source software package DBParser to combine and validate the search results from OMSSA, Mascot, and X!Tandem. DBParser produces combined or compared peptide or protein lists for subsequent evaluation by research scientists. Protein summaries are based upon the principle of parsimony, identifying the minimal set of proteins that can explain all of the observed peptides. The program generates html output reports of the sorted and compared peptide/protein lists which can be used for subsequent analysis. A CGI-based graphical user interface allows execution of DBParser over a network using a web browser to facilitate its use by multiple laboratories. DBParser has been refined to include quantification. A new technique to determine protein stoichiometry in macro-protein complexes has been tested and is being refined. Protein complexes isolated from mitochondria or sub-cellular organelles are being explored to determine the best methods of isolation and analysis. A new fluorescence detector designed and constructed at NIH is being tested with fluorescent peptide derivatives for quantification and comparison of complex mixtures.