Nanoacquity q tof ultima global instrumentation

Rhodococcus sp. AD45 cells grown on succinate or isoprene, for the proteomic analysis shown in Fig. 3, were broken by three passages through a French pressure cell (American Instrument) at 110 MPa (on ice). Cell debris was removed by centrifugation (10 000 g, 15 min, 4°C). Proteins were separated by SDS-PAGE, and bands of interest were excised from the gel for the identification of polypeptides by the Biological Mass Spectrometry and Proteomics Facility in the School of Life Sciences, University of Warwick, UK. Coomassie Brilliant Blue-stained gel pieces were processed and tryptically digested using the manufacturer’s recommended protocol on the MassPrep robotic protein handling system (Micromass, Manchester, UK). The extracted peptides from each sample were analysed by nano liquid-chromatography electrospray-ionization tandem mass spectrometry (LC-ESI-MS/MS) using NanoAcquity/Q-ToF Ultima Global instrumentation (Waters Corporation, Manchester, UK) with a 15 min liquid chromatography gradient. All MS and MS/MS data were corrected for mass drift using reference data collected from human [Glu¹]-fibrinopeptide B (catalogue F3261, Sigma). The data were used to interrogate a database compiled from predicted coding sequences of Rhodococcus sp. AD45 using the Waters ProteinLynx Global Server v2.5.1.

Protein bands of interest were excised from SDS-PAGE gels and tryptically digested using the manufacturer's recommended protocol on the MassPrep robotic protein handling system (Waters). The extracted peptides from each sample were analyzed by means of nanoLC-ESI-MS/MS using the NanoAcquity/Q-ToFUltima Global instrumentation (Waters) using a 45-min LC gradient. All MS data were corrected for mass drift using reference data collected from the [Glu¹]-Fibrinopeptide B (human—F3261 Sigma) sampled each minute of data collection. The data were then used to interrogate a database made up of the predicted protein sequences from RLP1 or RPP1 appended with the common Repository of Adventitious Proteins sequences (http://www.thegpm.org/cRAP/index.html) using ProteinLynx Global Server v2.3. All protein identification was carried out in the in-house Biological Mass Spectrometry and Proteomics Facility of the School of Life Sciences at the University of Warwick.