, 2009). Protein extract (20 μL) was mixed with solution UA (200 μL; 8 M urea in H2O, pH 8.5). This solution was loaded onto a 10-kDa

cut-off filter spin filter and centrifuged (14 000 g, 40 min). The retentate was washed three times with solution UA and the flow-through discarded. Then a solution of iodoacetamide (100 μL; 0.05 M in-solution UA) was added to the filter and incubated for 5 min. The filters were then centrifuged (14 000 g, 30 min) and washed twice with selleck compound a urea solution (100 μL; 8 M in H2O, pH 8.0). After each wash, the filter units were centrifuged (14 000 g; 40 min). Dimethyl labeling was performed essentially as described by Boersema et al. (2009). Briefly, the isolated proteins on the filter device were subjected to a Lys-C digestion. The resulting peptides were reconstituted in 100 mM TEAB buffer (Sigma, St. Louis, MO). Samples for ‘light’ labeling were mixed with formaldehyde (4% in H2O; Sigma). Samples for ‘heavy’ labeling were mixed with formaldehyde-D2 (4% in H2O; Sigma). Both samples were then mixed with freshly prepared sodium cyanoborohydride (0.6 M). After incubation for 1 h at room temperature, the reaction was quenched with ammonia

solution (1% v/v) and TFA. The acidified samples were desalted on StageTips made from C18 disks excised from Empore High Performance Extraction Disks (3M, St. Paul, MN) in a pipette tip (Rappsilber et al. 2007). Peptide mixtures were separated by find more nanoLC using an Agilent 1200 nanoflow system connected to either an LTQ Orbitrap XL or LTQ FT Ultra mass spectrometer (both from Thermo Electron, Bremen, Germany) equipped with a nanoelectrospray ion source (Proxeon Biosystem, Odense, Denmark). Chromatographic separation of the peptides took place in an in-house packed 20 cm fused silica emitter

(75-μm i.d.) with reverse-phase ReproSil-Pur C18-AQ (3 μm) resin (Maisch GmbH, Ammerbuch-Entringen, Germany). Peptides were injected onto the column (flow rate 500 nL min−1) and eluted with a flow of 250 nL min−1 from 5% to 40% acetonitril P-type ATPase in 0.5% acetic acid over 2 h. A ‘top 6’ acquisition method was set up on the mass spectrometer, utilizing the high mass accuracy of the Orbitrap for intact peptides and the speed and sensitivity of the LTQ (iontrap) for fragment spectra. The initial scan event was the intact peptide mass spectrum in the Orbitrap with range m/z 300–1800 and resolution R = 60 000 at m/z 400. Six CID fragmentation spectra in the iontrap (AGC target 5000, maximum injection time 150 ms) of the six most intense ions from the Orbitrap scan were recorded. Dynamic exclusion (2.5 min) and charge state screening requiring charge 2+ or more were enabled. The obtained tandem MS spectra were matched against theoretical spectra from a protein sequence database derived from the Cba. tepidum genome (GenBank acc. no. NC_002932) using Mascot (Matrix Science Ltd; www.matrixscience.com).