Preparation of samples for iTRAQ labeling and mass spectrometry

Flagellar samples were prepared for iTRAQ labeling as previously described (Bower et al., 2013 ) with minor modifications. Samples of whole flagella were washed briefly with 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, pH 7.4, centrifuged, and resuspended in dissolution buffer (0.5M triethylammonium bicarbonate, pH 8.5) for determination of protein concentration. Proteins were denatured and reduced, cysteine residues were blocked with methyl-methane thiosulfate, and then samples were digested overnight with trypsin. Aliquots of each sample (50 μg each) were reacted in duplicate with 4-plex iTRAQ labeling reagents (114–117; AB Sciex, Foster City, CA). After labeling, the samples were combined, vacuum dried, and purified with an Oasis MCX 1-cc Vac cartridge according to manufacturer’s instructions (Waters Corporation, Milford, MA) to remove excess trypsin, unreacted iTRAQ reagents, and buffer.

The iTRAQ-labeled sample was next fractionated offline using strong cation exchange (SCX) chromatography or high-pH, C18 reversed-phase chromatography. For SCX, the sample was resuspended in 20% (vol/vol) acetonitrile, 5 mM KH₂PO₄, pH 2.7, injected into a Magic 2002 HPLC system (Michrom BioResources, Auburn, CA) using a polysulfoethyl A column (PolyLC, Columbia, MD), and eluted with a gradient of 20% (vol/vol) acetonitrile, 5 mM KH₂PO₄, pH 3.0, and 500 mM KCl as previously described (Lund et al., 2007 ). For high-pH, C18 reversed-phase chromatography, the sample was resuspended in 10 mM ammonium formate, pH 10, in 98:2 water::acetonitrile, injected into a Magic 2002 HPLC system using a C18 Gemini-NX column (Phenomenex, Torrance, CA), and eluted with a gradient of 10 mM ammonium formate, pH 10, in 10:90 water:acetonitrile, as previously described (Tran et al. 2013 ). Peptide-containing fractions from the C18 column were divided into two equal-numbered groups, “early” and “late.” The first “early” fraction was concatenated with the first “late” fraction, and so on (Yang et al., 2012 ).

Fractions from both columns were vacuum dried, resuspended in load solvent (98:2:0.01, water:acetonitrile:formic acid), and loaded in 1- to 1.5-μg aliquots for capillary LC using a C18 column at low pH. The C18 column was mounted in a nanospray source directly in-line with a Velos Orbitrap mass spectrometer (Thermo Fisher Scientific, Waltham, MA). Online capillary LC and tandem mass spectrometry were performed as previously described (Lin-Moshier et al., 2013 ), with the exception that the higher-energy, collision-induced dissociation activation time was 20 instead of 0.1 ms because iTRAQ-labeled peptides need more energy to dissociate reporter ions.