Journal
JOURNAL OF PROTEOME RESEARCH
Volume 7, Issue 3, Pages 960-968Publisher
AMER CHEMICAL SOC
DOI: 10.1021/pr070432+
Keywords
monkeypox; vaccinia; proteomics; mass spectrometry; SEQUEST; X! Tandem
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Funding
- NCRR NIH HHS [P41 RR018522-05, P41 RR018522, RR18522] Funding Source: Medline
- NIAID NIH HHS [Y1-AI-4894-01] Funding Source: Medline
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Orthopoxviruses are among the largest and most complex of the animal viruses. In response to the recent emergence of monkeypox in Africa and the threat of smallpox bioterrorism, two orthopoxviruses with different pathogenic potentials, human monkeypox virus and vaccinia virus, were proteomically compared with the goal of identifying proteins required for pathogenesis. Orthopoxviruses were grown in HeLa cells to two different viral forms (intracellular mature virus and extracellular enveloped virus), purified by sucrose gradient ultracentrifugation, denatured using RapiGest surfactant, and digested with trypsin. Unfractionated samples and strong cation exchange HPLC fractions were analyzed by high-resolution reversed-phase nano-LC-MS/MS, and analyses of the MS/MS spectra using SEQUEST and X! Tandem resulted in the confident identification of hundreds of monkeypox, vaccinia, and copurified host-cell proteins. The unfractionated samples were additionally analyzed by LC-MS using an LTQ-Orbitrap, and the accurate mass and elution time tag approach was used to perform quantitative comparisons. Possible pathophysiological roles of differentially abundant Orthopoxvirus proteins are discussed. Data, processed results, and protocols are available at http://www.proteomicsresource.org/.
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