Maxent

All complexes were analyzed using SYNAPT G2-Si High Definition Mass Spectrometer (Waters, Manchester, U.K.). For the analysis, all protein solutions were buffer exchanged into 200 mM ammonium acetate (pH 7.5) using Micro Bio-Spin chromatography columns (Bio-Rad). Aliquots (∼2 μL) were introduced into the mass spectrometer via nanoflow capillaries using the following conditions: capillary voltage 1.2 kV, sampling cone 120 V, source offset 20 V. The source temperature was set up for 25 °C. The collision voltage was adjusted for the optimal signal level. Maximum entropy (MaxEnt, Waters) deconvolution was applied to electrospray data to recalculate the gas phase existing masses.

Mass spectrometry studies of intact flagellin proteins were carried out using 1 μg or less of protein, as described in our previous work (Wilhelms et al., 2012 (link)). Briefly, purified flagellin samples were injected onto a protein microtrap (Michrom Bioresources Inc., Auburn, CA, USA) connected to a gradient HPLC pump (Agilent 1100 HPLC). To resolve the proteins, a gradient of 5–60% solvent B (1 mL/min) over 60 min was used, where Solvent A was 0.1% formic acid in HPLC grade water and solvent B was 0.1% formic acid in acetonitrile. A precolumn splitter was used to direct ∼35 μl/min of the HPLC mobile phase through the trap or column and into the electrospray interface of the QTOF2 (Waters, Milford, MA, USA) or Orbitrap XL Mass Spectrometer (Thermal, CA, USA) to allow real-time monitoring of ion elution profiles. Intact masses of proteins were calculated using MaxEnt (Waters, Beverly, MA, USA) software by spectral deconvolution.
To identify potential glycopeptides, flagellin (50–200 μg) was digested and analyzed as previously described (Wilhelms et al., 2012 (link)). Unmodified peptides were identified using MASCOT (Matrix Science, London, UK) as described (Wilhelms et al., 2012 (link)). Glycopeptide MS/MS spectra were de novo sequenced as previously described (Wilhelms et al., 2012 (link)).