Croft Nathan P, de Verteuil Danielle A, Smith Stewart A, Wong Yik Chun, Schittenhelm Ralf B, Tscharke David C, Purcell Anthony W
From the ‡Department of Biochemistry and Molecular Biology, Monash University, Clayton, VIC, 3141, Australia;
§Research School of Biology, The Australian National University, Canberra, ACT, 0200, Australia.
Mol Cell Proteomics. 2015 May;14(5):1361-72. doi: 10.1074/mcp.M114.047373. Epub 2015 Mar 9.
The generation of antigen-specific reagents is a significant bottleneck in the study of complex pathogens that express many hundreds to thousands of different proteins or to emerging or new strains of viruses that display potential pandemic qualities and therefore require rapid investigation. In these instances the development of antibodies for example can be prohibitively expensive to cover the full pathogen proteome, or the lead time may be unacceptably long in urgent cases where new highly pathogenic viral strains may emerge. Because genomic information on such pathogens can be rapidly acquired this opens up avenues using mass spectrometric approaches to study pathogen antigen expression, host responses and for screening the utility of therapeutics. In particular, data-independent acquisition (DIA) modalities on high-resolution mass spectrometers generate spectral information on all components of a complex sample providing depth of coverage hitherto only seen in genomic deep sequencing. The spectral information generated by DIA can be iteratively interrogated for potentially any protein of interest providing both evidence of protein expression and quantitation. Here we apply a solely DIA mass spectrometry based methodology to profile the viral antigen expression in cells infected with vaccinia virus up to 9 h post infection without the need for antigen specific antibodies or other reagents. We demonstrate deep coverage of the vaccinia virus proteome using a SWATH-MS acquisition approach, extracting quantitative kinetics of 100 virus proteins within a single experiment. The results highlight the complexity of vaccinia protein expression, complementing what is known at the transcriptomic level, and provide a valuable resource and technique for future studies of viral infection and replication kinetics. Furthermore, they highlight the utility of DIA and mass spectrometry in the dissection of host-pathogen interactions.
对于表达成百上千种不同蛋白质的复杂病原体或具有潜在大流行特性、因此需要快速研究的新兴病毒株或新病毒株的研究而言，抗原特异性试剂的生成是一个重大瓶颈。在这些情况下，例如，要覆盖整个病原体蛋白质组，开发抗体的成本可能高得令人望而却步，或者在可能出现新的高致病性病毒株的紧急情况下，交付时间可能长得令人无法接受。由于可以快速获取此类病原体的基因组信息，这为使用质谱方法研究病原体抗原表达、宿主反应以及筛选治疗药物的效用开辟了途径。特别是，高分辨率质谱仪上的数据非依赖型采集（DIA）模式可生成复杂样品所有成分的光谱信息，提供了迄今仅在基因组深度测序中才有的覆盖深度。DIA生成的光谱信息可以反复查询任何感兴趣的蛋白质，既提供了蛋白质表达的证据，也提供了定量信息。在这里，我们应用一种完全基于DIA质谱的方法，在感染痘苗病毒后长达9小时的细胞中分析病毒抗原表达，而无需抗原特异性抗体或其他试剂。我们使用SWATH-MS采集方法展示了对痘苗病毒蛋白质组的深度覆盖，在单个实验中提取了100种病毒蛋白的定量动力学。结果突出了痘苗蛋白表达的复杂性，补充了转录组水平上已知的信息，并为未来病毒感染和复制动力学研究提供了宝贵的资源和技术。此外，它们突出了DIA和质谱在剖析宿主-病原体相互作用方面的效用。
