Massprep liquid handling robot

The LS from B. amyloliquefaciens was purified by size-exclusion chromatography and was separated by SDS-PAGE gel. The LS bands were removed and sent for proteomic analysis by mass spectrometry at the Plateforme de Protéomique—Centre de Recherche du CHU de Québec (Laval, Québec, Canada). The bands were subjected to tryptic digestion using a MassPrep liquid handling robot (Waters) following the protocol by Brotherton et al. [45 (link)]. After an initial reduction (10 mM dithiothreitol) and alkylation (55 mM iodoacetamide), the sample was digested with using 126 nM porcine trypsin (sequencing grade, Promega) at 58 °C for 1 h. The digestion products were extracted using 1% formic acid and 2% acetonitrile, which was then followed by 1% formic acid and 50% acetonitrile. The protein extracts were pooled, dried by vacuum centrifugation, and then resuspended into 10 µL of 0.1% formic acid for analysis by electrospray ionization mass spectrometry. The peptide samples were separated by an online reverse-phase (RP) nanoscale capillary liquid chromatography (nanoLC) and analyzed by electrospray mass spectrometry (ESI MS/MS) [46 (link)]. The fragments were analyzed using Scaffold software (version 4.0) (Proeome Software).

Bands of interest were extracted from gels, placed in 96-well plates, and washed with water. Tryptic digestion was performed using a MassPrep liquid handling robot (Waters, Milford, USA) according to the manufacturer’s specifications, and the protocol of Shevchenko et al. [25 (link)] was followed with the modifications suggested by Havlis et al. [26 (link)]. Briefly, proteins were reduced with 10 mM DTT and alkylated with 55 mM iodoacetamide. Trypsin digestion was performed using 126 nM modified porcine trypsin (sequencing grade, Promega, Madison, WI, USA) at 58°C for 1 h. The digestion products were extracted using 1% formic acid and 2% acetonitrile followed by 1% formic acid and 50% acetonitrile. The recovered extracts were pooled, vacuum centrifuge-dried and subsequently resuspended in 10 µL of 0.1% formic acid. Aliquots (2 µL) of the extracts were then analyzed by mass spectrometry (MS).