After separation of DIGE-labeled strips by SDS-PAGE, gels were scanned in the glass plates using a three laser Typhoon 9400 variable mode imager (GE Healthcare, Piscataway, NJ) at 200 microns. Differences in protein spots were

quantified using DeCyder 2-D Differential Analysis Software v7.2. Protein spots of interest were excised and processed for mass spectrometry as previously described [49]. Dried peptides were sent to the Protein Chemistry section of the NIAID Research Technologies Branch, NIH for identification as described below. The recovered peptides were re-suspended in 5 ul of Solvent A (0.1% formic acid, 2% acetonitrile, and 97.9% water). Prior to mass spectrometry analysis, the re-suspended peptides were chromatographed directly on column, without trap clean-up. The bound peptides were separated at 500 nl/min generating 80–120 Bar pressure, selleck chemicals llc using an AQ C18 reverse phase media (3 u particle size and

200 u pore) packed in a pulled tip, nano-chromatography column (0.100 mm ID × 150 mm L) from Precision Capillary Columns, San Clemente, CA. The chromatography was performed in-line with an LTQ-Velos Orbitrap mass spectrometer (ThermoFisher Scientific, West Palm Beach, FL) and the mobile phase consisted of a linear gradient prepared from solvent A and solvent B (0.1% formic acid, 2% water, and 97.9% acetonitrile) at room temperature. Nano LC-MS (LC-MS/MS) was learn more performed with a ProXeon Easy-nLC II multi-dimensional liquid chromatograph and temperature controlled Ion Max Nanospray source (ThermoFisher Scientific) in-line with the LTQ-Velos Orbitrap mass spectrometer. Mass calibration was performed as needed with the positive ion Cal Mix prepared as described by Thermo-Scientific and monitored by routine analysis of a 10 femtomole stock sample of BSA digest. Typical acceptable results

for this analysis would yield a 2800 – 3300 Mascot score, 75 – 85% coverage and 0 – +/−4 ppm error when submitted to the Mascot server using Proteome Discoverer 1.3 using the Swiss Prot-Trembl data base. Computer controlled data dependent automated switching to MS/MS by Xcalibur 2.1 software was used for data acquisition Urocanase and provided the peptide sequence information. Data processing and databank searching were performed with PD 1.3 and Mascot software (Matrix Science, Beachwood, OH). Acknowledgements The authors gratefully acknowledge the generous gifts of strains and advice from David Haake and Marije Pinne. We also thank Joe Hinnebusch and Frank Gherardini for critical reading of the manuscript; Dan Sturdevant, Kevin Lawrence and Julie Boylan for technical advice and helpful discussions, Jeff www.selleckchem.com/products/q-vd-oph.html Skinner at Bioinformatics and Computational Biosciences Branch for statistical analysis, and Scott Samuels’ lab for technical advice on RNA isolation. This research was supported by the Intramural Research Program of the NIH, NIAID. Electronic supplementary material Additional file 1: Distribution of bat genes in the Spirochaetes.