| Control of infectious diseases requires
methods for the rapid and accurate detection of the respective
causative agents. However, currently this need is not
always met, as illustrated by the recent recurrent outbreaks
of Norovirus-related gastrointestinal diseases on cruise
ships and by the present multi-country outbreak of unexplained
atypical pneumonia, known as severe acute respiratory
syndrome (SARS). When developing novel methods for pathogen
detection and identification, the capsid proteins of human
viruses represent excellent diagnostic targets because
of (i) their relatively high abundance within the virus
particle, (ii) their characteristic molecular masses,
peptide sequences and digestion fragments, and (iii) their
amenability to detection by matrix-assisted laser desorption/ionization
time-of-flight mass spectrometry (MALDI-TOF MS), one of
the fastest and most robust tools in mass spectrometry
available today.
In this study, we explored the possibility of detecting
the characteristic 56 kDa Norwalk virus (NV) capsid protein
by MALDI-TOF MS in simulated clinical samples mimicking
highly purified extracts of virus-containing human and/or
environmental specimens. Suspensions containing NV-like
particles were evaluated. The bacteriophage MS2 was added
at varying ratios to simulate the presence of co-extracted
viral interferences. Aliquots of samples containing atto-mole
to femto-mole quantities of NV capsid protein were processed
and analyzed by MALDI-TOF MS. Samples (15 µL aliquots)
were placed into a 0.5 mL microcentrifuge tube, amended
with 250 ng of porcine trypsin and incubated at 37°C
(5 min to 18 hours). Then, samples were dried down in
a speed vac, cleaned up with Millipore C-18 Zip Tips and
analyzed by MALDI-TOF MS. Approximately 25% of the sample
volume was spotted onto a target plate holding an equal
volume of 2,5-dihydroxybenzoic acid (DHB) matrix. Samples
were analyzed with a Voyager DE-STR MALDI-TOF mass spectrometer
running in reflector mode for positive ions using external
calibration. The obtained mass spectra of the digest were
analyzed using standard protein database searches (NCBI,
etc.). Score results for highly dilute samples and for
various binary mixtures of viral particles clearly demonstrated
that the method can successfully detect the target at
clinically relevant concentrations. This study represents
the first report on the detection of NV capsid by using
MALDI-TOF MS.
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