Ultimate 3000 rapid separation lc

Purified GIIα was subjected to SDS-PAGE, Coomassie protein staining and the band of interest (~100 kDa) was excised from the gel and dehydrated using acetonitrile and vacuum centrifugation. Dried gel pieces were reduced with 10 mM dithiothreitol, alkylated with 55 mM iodoacetamide and gel pieces were then washed alternately with 25 mM ammonium bicarbonate followed by acetonitrile. This was repeated, and the gel pieces dried by vacuum centrifugation prior to digestion with trypsin for 16 h at 37°C. Digested samples were analyzed by LC-MS/MS using an UltiMate^® 3000 Rapid Separation LC (RSLC, Dionex Corporation, Sunnyvale, CA) coupled to an Orbitrap Elite (Thermo Fisher Scientific, Waltham, MA) mass spectrometer. Peptide mixtures were separated using a gradient from 92% A (0.1% FA in water) and 8% B (0.1% FA in acetonitrile) to 33% B, in 44 min at 300 nL min⁻¹, using a 75 mm × 250 μm i.d. 1.7 mM BEH C18, analytical column (Waters). Peptides were selected for fragmentation automatically by data dependent analysis. Data produced were searched using Mascot (Matrix Science UK), against the amino acid sequence of GIIα (Supplementary Figure S5) and the Swissprot and Trembl database with taxonomy of E. coli selected. Data were validated using Scaffold (Proteome Software, Portland, OR).

LC-MS/MS was performed by the Biological Mass Spectrometry Core Facility in the Faculty of Biology, Medicine and Health at the University of Manchester (Manchester, UK). As previously described in their protocols [21 (link),101 (link),102 (link)]: vacuum dried peptide samples were analysed via LC-MS/MS using an UltiMate® 3000 Rapid Separation LC (Dionex Corp; Sunnyvale, CA, USA) and an Orbitrap Elite mass spectrometer (Thermo Fisher Scientific). Peptide mixtures were separated using a 250 mm × 75 μm i.d. 1.7 mM BEH C18, analytical column (Waters, Hertfordshire, UK) on a gradient of 92% A (0.1% [v/v] formic acid in water) and 8% B (0.1% [v/v] formic acid in acetonitrile) to 33% B. These were run for 60 min with a flow rate of 300 nl/min. Peptides were automatically picked for fragmentation via data-dependent analysis.

LC-MS/MS were carried out using an UltiMate^® 3000 Rapid Separation LC (RSLC, Dionex Corporation, Sunnyvale, CA, USA) coupled to an Orbitrap Elite (Thermo Fisher Scientific, Waltham, MA, USA) mass spectrometer. Peptide mixtures were separated using a gradient from 92% A (0.1% FA in water) and 8% B (0.1% FA in acetonitrile) to 33% B in 44 min at 300 nL min⁻¹ using a 250 mm × 75 μm i.d. 1.7 μM M-Class CSH C18 analytical column (Waters). Peptides were selected for fragmentation automatically by data-dependent analysis, with the top 6 ions selected per cycle. Fragmentation was achieved via CID. The m/z range for precursors was 350–1500 with a normalized collision energy of 35. The minimum signal threshold was 500 counts and an isolation width of 1 Da. All charge states were allowed except singly charged and unassigned charge states. Precursor ions were selected for fragmentation twice, with an exclusion window of 30 s. Precursor ions were measured in the Orbitrap with a resolution of 120,000 in profile.