Protein spot picking, digestion and identification was performed at the Proteomics Platform of the CRP Gabriel Lippmann in Luxembourg. Spot handling and digestion was performed on an Ettan Spot Handling Workstation (GE Healthcare). Spots of 2 mm in diameter were picked and washed twice in 100 µl 50 mM ammonium bicarbonate (Sigma-Aldrich)/50% methanol (Biosolve, Valkenswaard, Nederland) for 20 min and 30 min respectively at room temperature, dehydrated in 2 steps of 30 min in 80 µl 75% ACN (Biosolve) at room temperature and dried at 40 °C for 20 min. Gel spot rehydration and protein digestion was performed in 8 µl of 20 mM ammonium bicarbonate supplemented with 5 ng/µl trypsin (Promega, Mannheim, Germany) for 6 h at 37 °C. Peptides were extracted from the picked gel plugs with 50% ACN/0.1% TFA (Sigma-Aldrich) and dried for 20 min at 37 °C. Peptides were redissolved in 3 µl of 50% ACN/ 0.1% TFA and 0.7 µl were spotted on the 384 target (Bruker, Bremen, Germany) and mixed with 0.7 µl of a matrix containing 7 mg/ml a-cyano-4-hydrocinnamic acid (Bruker) /50% ACN/0.1% TFA. Peptides were then analyzed on a 4800 MALDI-TOF/TOF (Life Technologies) mass spectrometer. MS and MS/MS were calibrated using trypsin fragments (1798.857, 1940.95, 2211.105Da) and the 4700 calibration mix (Applied Biosystems), respectively. For MS analysis, the mass range was set from 800-4000Da and a minimal signal/noise ratio to 10. A peak density filter of 50 peaks/200Da and a maximum of 65 peaks were used for identification. Trypsin fragments (842.4, 1940.92, 2211.10, 2233.16, 1045.55, 2299.18, 1707.77, 1944.92, 2011.98) were excluded. For MS/MS analysis, the mass range was chosen 20-60Da below the respective precursor mass. The other parameters were identical to MS analysis. GPS explorer V3.6 (Applied Biosystems) was used for peak picking and MASCOT v2.1 (Matrix Science Ltd., London, GB) was used for database search in SwissProt (database release September 2010 containing 519,348 sequences), setting the taxonomy to human (20,359 sequences), a maximum of 2 missed cleavages, variable and fixed modifications to methionine oxidation and cysteine carbamidomethylation respectively, precursor ion tolerance to 150 ppm and MS/MS fragment tolerance to 0.75 Da. The false discovery rate was set at 5% and MASCOT scores above 56 were considered significant. Representative gel pictures with identified spots and the peak lists used for identification with MASCOT are available at the Swiss 2D-PAGE repository. Data were submitted to the Ingenuity Pathway Analysis (IPA) (Ingenuity Systems, www.ingenuity.com).