Department for Bioanalysis and Horizon Technologies, Health Protection Agency, 61 Colindale Avenue, London NW95EQ, United Kingdom.
J Microbiol Methods. 2012 Feb;88(2):237-47. doi: 10.1016/j.mimet.2011.12.001. Epub 2011 Dec 9.
Bacillus anthracis is the causative agent of anthrax, an acute and often fatal disease in humans. Due to the high genomic relatedness within the Bacillus cereus group of species it is a challenge to identify B. anthracis consistently. Alternative strategies such as proteomics coupled with mass spectrometry (MS) provide a powerful approach for biomarker discovery. However, validating and evaluating these markers, particularly for genetically homogeneous species such as B. anthracis are challenging. The objective of this study is to develop a robust biomarker discovery and validation pipeline, using proteomic methodology combined with in silico and molecular approaches, to determine a biomarker list, using B. anthracis as a model. In this exploratory study we profiled the proteome of B. anthracis and genetically related species using GeLC-Liquid Chromatography MS/MS (GeLC-LC MS/MS), identifying peptides that could be used to detect B. anthracis. Peptides were filtered to remove low quality identifications. Using comparative bioinformatic approaches, matching and searching against genomic sequence data a shortlist of peptide biomarkers was determined and validated using DNA sequencing, against a panel of closely related strains, to determine marker specificity. Further validation was performed using MS quantitation methods to assess sensitivity and specificity. A biomarker discovery pipeline was successfully developed in this study, comprising four distinct stages: proteome profiling, comparative bioinformatic validation, DNA sequencing and MS validation. Using the pipeline, 5379 peptides specific for Bacillus species and 36 peptides specific for B. anthracis were identified and validated. The 36 peptides, representing 30 proteins were derived from over 15 different clusters of orthologous group categories, including proteins involved in transcription, energy production/conservation as well as multifunctional proteins. We demonstrated that the peptide biomarkers identified in this study could be detected in a complex background, in which 0.1 μg of protein extract from B. anthracis was spiked into 9.90 μg of B. cereus protein extracts. The integration of both stable non-redundant peptides with molecular methodology for marker discovery and validation, improves the robustness of identifying and characterising candidate biomarkers for the identification of bacteria such as B. anthracis.
炭疽杆菌是炭疽病的病原体，炭疽病是一种人类急性且常致命的疾病。由于芽孢杆菌属内的种具有高度的基因组相关性，因此一致地鉴定炭疽杆菌具有挑战性。替代策略，如蛋白质组学与质谱（MS）相结合，为生物标志物的发现提供了一种强大的方法。然而，验证和评估这些标记物，特别是对于遗传上同质的物种，如炭疽杆菌，具有挑战性。本研究的目的是开发一种稳健的生物标志物发现和验证管道，使用蛋白质组学方法结合计算机和分子方法，使用炭疽杆菌作为模型，确定一个生物标志物列表。在这项探索性研究中，我们使用凝胶电泳-液相色谱-MS/MS（GeLC-LC MS/MS）对炭疽杆菌和遗传上相关的物种进行了蛋白质组学分析，鉴定出了可用于检测炭疽杆菌的肽段。对肽段进行了过滤，以去除低质量的鉴定结果。使用比较生物信息学方法，根据基因组序列数据进行匹配和搜索，确定了一个短肽生物标志物列表，并使用 DNA 测序对一组密切相关的菌株进行验证，以确定标记物的特异性。进一步使用 MS 定量方法进行验证，以评估灵敏度和特异性。本研究成功开发了一种生物标志物发现管道，该管道由四个不同的阶段组成：蛋白质组学分析、比较生物信息学验证、DNA 测序和 MS 验证。使用该管道，鉴定并验证了 5379 种特定于芽孢杆菌属的肽段和 36 种特定于炭疽杆菌的肽段。这 36 种肽段代表 30 种蛋白质，来源于 15 个以上不同的直系同源群类别簇，包括参与转录、能量产生/消耗以及多功能蛋白的蛋白质。我们证明，在复杂背景下可以检测到本研究中鉴定的肽类生物标志物，其中 0.1μg 的炭疽杆菌蛋白提取物与 9.90μg 的蜡样芽胞杆菌蛋白提取物混合。将稳定的非冗余肽段与用于标志物发现和验证的分子方法相结合，提高了鉴定和表征候选生物标志物的稳健性，从而鉴定和表征炭疽杆菌等细菌。
