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Applied and Environmental Microbiology, April 2006, p. 2749-2755, Vol. 72, No. 4
0099-2240/06/$08.00+0     doi:10.1128/AEM.72.4.2749-2755.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Detection of Norovirus Capsid Protein in Authentic Standards and in Stool Extracts by Matrix-Assisted Laser Desorption Ionization and Nanospray Mass Spectrometry

David R. Colquhoun,1 Kellogg J. Schwab,1,2 Robert N. Cole,3 and Rolf U. Halden1,2*

Center for Water and Health, Department of Environmental Health Sciences, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, Maryland 21205,1 Center for Public Health Preparedness, Department of Environmental Health Sciences, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, Maryland 21205,2 Mass Spectrometry and Proteomics Facility, Institute for Basic Biomedical Sciences, School of Medicine, Johns Hopkins University, Baltimore, Maryland 212053

Received 19 December 2005/ Accepted 10 February 2006

Mass spectrometry (MS) represents a rapid technique for the identification of microbial monocultures, and its adaptation to the detection of pathogens in real-world samples is a public health and homeland security priority. Norovirus, a leading cause of gastroenteritis in the world, is difficult to monitor because it cannot be cultured outside the human body. The detection of norovirus capsid protein was explored using three common MS-based methods: scanning of intact proteins, peptide mass fingerprinting, and peptide sequencing. Detection of intact target protein was limited by poor selectivity and sensitivity. Detection of up to 16 target peptides by peptide mass fingerprinting allowed for the reproducible and confident (P < 0.05) detection of the 56-kDa norovirus capsid protein in the range of 0.1 x 10–12 to 50 x 10–12 mol in authentic standards of recombinant norovirus virus-like particles (VLPs). To explore assay performance in complex matrixes, a non-gel-based, rapid method (2 to 3 h) for virus extraction from human stool was evaluated (72% ± 12% recovery), and additional analyses were performed on norovirus-free stool extracts fortified with VLPs. Whereas peptide mass fingerprinting was rendered impractical by sample interferences, peptide sequencing using nanospray tandem MS facilitated unambiguous identification of ≥250 fmol of capsid protein in stool extracts. This is the first report on MS-based detection of norovirus, accomplished by using structurally identical, noninfective VLPs at clinically relevant concentrations. It represents an important milestone in the development of assays for surveillance of this category B bioterrorism agent.

* Corresponding author. Mailing address: JHU Center for Water and Health, Department of Environmental Health Sciences, Bloomberg School of Public Health, Johns Hopkins University, BSPH Bldg., Room E6618, Baltimore, MD 21205. Phone: (410) 955-2609. Fax: (410) 955-9334. E-mail: rhalden{at}jhsph.edu.

Applied and Environmental Microbiology, April 2006, p. 2749-2755, Vol. 72, No. 4
0099-2240/06/$08.00+0     doi:10.1128/AEM.72.4.2749-2755.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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