Novel Applications of Mass Spectrometry and Proteomics to Infectious Disease Diagnostics

质谱和蛋白质组学在传染病诊断中的新应用

• 批准号: 9549512
• 负责人: John Dekker
• 金额: --
• 依托单位: CLINICAL CENTER
• 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/9549512
• 关键词: Acinetobacter baumannii; Antimicrobial Resistance; Bacteria; Bacterial Proteins; Biological Assay; Classification; Clinical; Clinical Chemistry; Clinical Microbiology; Collaborations; Communicable Diseases; Computer Analysis; Computer Simulation; Development; Diagnostic; Epidemiology; Genes; Genome; Goals; Laboratories; MALDI-TOF Mass Spectrometry; Mass Spectrum Analysis; Methods; Modeling; Molecular; Multi-Drug Resistance; Organism; Peptides; Plasmids; Proteins; Proteomics; Public Health; Publishing; Reporting; Resistance; Specimen; Techniques; Technology; Testing; Time; United States National Institutes of Health; Work; assay development; bacterial resistance; base; carbapenemase; design; novel; pathogen; pathogenic bacteria; patient oriented; protein biomarkers; resistance mechanism; validation studies

Rapid molecular methods for identification of pathogens and characterization of resistance mechanisms are essential to clinical infectious disease diagnostics. Our work aims to develop novel applications of mass spectrometry to these problems in the clinical microbiology laboratory. Our approach involves both the identification of new bacterial protein markers and the development of assays to detect these markers using LC-MS/MS and other mass spectrometry technologies. Methods are being pursued for (1) Culture-free identification of pathogenic bacteria in primary specimens, (2) Strain-level classification of bacterial pathogens, and (3) Rapid resistance protein identification. Previously published work involved identification of a peptide marker for tracking a carbapenemase-carrying resistance plasmid that could be detected by MALDI-TOF mass spectrometry (Lau et al, JCM, 2014), followed by a clinical validation study demonstrating the method's utility in identifying KPC carbapenemase proteins in clinical isolates from NIH Clinical Center patients (Youn et al, JCM, 2016). Work done during the 2016 fiscal year in collaboration with Anthony Suffredini's group in CCMD, NIHCC included the development of a genoproteomics approach for identifying strain-specific peptide markers based on LC-MS/MS profiling of tryptic peptides, using clinical Acinetobacter baumannii isolates as a model (Wang et al, Clinical Chemistry, 2016). This approach is based on in silico computational analysis to guide selection of informative, genome-specific tryptic peptides from LC-MS/MS peptide profiles, followed by experimental confirmation. Work completed during the past fiscal year included extension of genoproteomic methods to the design of assays for the culture-free identification of bacteria in primary specimens, published in Wang et al, Clinical Chemistry, 2017. This proof-of-concept study demonstrated that rapid (90 minute) diagnostic identification of bacterial pathogens in primary specimens may be possible for a variety of specimen types without culture. The genoproteomic approach was also extended to the direct identification of the KPC carbapenemase in clinical isolates using specific tryptic peptides that can be detected by LC-MS/MS, published in Wang et al, Scientific Reports, 2017. This method allows a rapid (90 minute) diagnostic mass spectrometry assay for detecting the KPC carbapenemase protein in clinical isolates. Work is currently underway to extend culture free identification methods to a variety of high impact pathogenic bacteria. Additionally, work is underway to develop LC-MS/MS assays to detect other carbapenemases and extended spectrum beta lactamses using tryptic peptides and LC-MS/MS, which may be used in multiplex.

用于鉴定病原体的快速分子方法和抵抗机制的表征对于临床传染病诊断至关重要。我们的工作旨在开发质谱法对临床微生物学实验室中这些问题的新颖应用。我们的方法涉及使用新细菌蛋白标记物的鉴定，也涉及使用LC-MS/MS和其他质谱技术检测这些标记的测定方法的开发。 （1）在原发性标本中对病原细菌的无培养鉴定，（2）细菌病原体的应变水平分类以及（3）快速抗性蛋白鉴定。 Previously published work involved identification of a peptide marker for tracking a carbapenemase-carrying resistance plasmid that could be detected by MALDI-TOF mass spectrometry (Lau et al, JCM, 2014), followed by a clinical validation study demonstrating the method's utility in identifying KPC carbapenemase proteins in clinical isolates from NIH Clinical Center patients (Youn et al, JCM, 2016). NIHCC与Anthony Suffredini的小组合作，在2016财政年度完成的工作包括开发一种基于LC-MS/MS的胰蛋白酶/MS分析，使用临床上的Acinetabacter baumantabter baumannii Inalike As Model As Model Alical Clinatial Chanical Chanical，开发了一种基于LC-MS/MS的菌株特异性肽标记的方法。这种方法基于计算机计算分析，以指导从LC-MS/MS肽谱的信息，基因组特异性胰蛋白酶肽的选择，然后进行实验确认。 在上一个财政年度完成的工作包括将基因蛋白质组学方法扩展到设计的测定方法，以设计基本标本中细菌的无培养物识别，该测量鉴定在Wang等人，临床化学，2017年。这项概念证明的研究表明，在不使用类型培养的初级表明中，可以在初级表现中快速（90分钟）对细菌病原体的诊断识别。 The genoproteomic approach was also extended to the direct identification of the KPC carbapenemase in clinical isolates using specific tryptic peptides that can be detected by LC-MS/MS, published in Wang et al, Scientific Reports, 2017. This method allows a rapid (90 minute) diagnostic mass spectrometry assay for detecting the KPC carbapenemase protein in clinical isolates. 目前正在进行工作，将自由鉴定方法扩展到各种高影响致病细菌。 此外，还正在进行开发LC-MS/MS分析的工作，以检测其他使用胰蛋白酶肽和LC-MS/MS的其他碳纤维苯甲酶和扩展的光谱βlactames，这可以在多路复用中使用。