Abstract
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.
Highlights
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
Systems biology strives to provide a holistic understanding of biological processes such as signaling pathways, gene expression, protein-protein interactions and changes induced by environmental stimuli or microorganisms [1]
SWATH-MS Profiles Viral Antigen Expression During Infection in a Single LC-MS Analysis—We have previously studied the kinetics of vaccinia virus (VACV) CD8ϩ T-cell epitope presentation in the murine C57BL/6-derived dendritic-like cell line DC2.4 and in the same study tracked the expression of eight VACV antigens by multiple reaction monitoring (MRM) [26]
Summary
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. Following peptide sequence input (e.g. through direct insertion or in silico digestion of a protein), SWATH-MS data can be searched rapidly for co-eluting precursor and product ions yielding XIC traces much like those generated by MRM experiments.
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