Dl dtt

The capsid protein subunits (L and S) of CPMV virus particles from Cowpea and CPMV eVLPs were separated on an SDS-PAGE gel as previously described (Sainsbury et al., 2011 (link)). The S subunit of CPMV virions and that of eVLPs contained two bands corresponding to the slow and fast forms (Figures 5A and 5B). The bands corresponding to the slow and fast forms of the S subunit of eVLPs and of the fast form (only) of the virions were excised and submitted for proteomics mass spectrometry analysis by digestion with GluC protease. The excised gel bands were destained with a mixture of a 50:50 (v/v) dilution of acetonitrile-25 mM ammonium bicarbonate and dried with a SpeedVac system. The protein samples in the gel were then reduced in 25 μl of 10 mM d,l-DTT (Sigma) for 1 hr and alkylated with 25 μl of 55 mM iodoacetamide (Sigma) for 30 min in the dark prior to an 18-hr GluC digestion at 37°C using a 1:30 (w/w) enzyme-to-substrate ratio. The resulting peptides were extracted twice with a 50:45:5 (v/v) volume of acetonitrile-water-formic acid and concentrated to 30 μl before being analyzed. Peptides were analyzed by reverse-phase chromatography prior to mass spectrometry analysis and tandem mass spectrometry samples were analyzed by using Mascot (version 2.1.04; Matrix Science) as described in Powers et al. (2011) (link).