Reduced using 0.5 l of reduction solution (TCEP-HCl, tris(2-carboxethy1)phosphine, 200 mM) and alkylated using 0.5 L of 400 mM iodoacetamide (IAA). To label the N-terminus and free amino groups at the lysine residues 3 L of C12or C 13 or D2 - nicotinoyloxysuccinimide (Nic-reagent) were added to each mixture and incubated for 3 h in the dark. Thereafter all three ICPL labeled samples wereJochim et al. Proteome Science 2011, 9:48 http://www.proteomesci.com/content/9/1/Page 10 ofcombined and proteins were precipitated by acetone to remove excess labeling reagent (as described above). Proteins were solved in Laemmli sample buffer, separated by gradient SDS-PAGE (8.5 -18 ) and stained with Coomassie brilliant blue. The gel lane was cut into 20-25 pieces PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/6388955 and in-gel digestion was performed; proteins were destained with 50 mM NH4HCO3 in 50 (v/v) acetonitrile (ACN) [50]. Then the gel pieces were dried with 100 ACN, vacuum evaporated and rehydrated with 20 L of 5 ng/L modified trypsin (Promega) in 20 mM NH4 HCO3 , 10 ACN. After incubation on ice for 1 h, residual trypsin solution was removed and substituted by 20 mM NH4HCO3 in 10 ACN. The digestion was performed over night at 37 . Supernatants were collected and the gel pieces were washed with 50 ACN in 0.2 (v/v) TFA and then dried in 10 (v/v) ACN. All supernatant containing peptides were pooled and dried. The dried peptide samples were stored at -80 until analysis. For separation by high performance liquid chromatography (HPLC) samples were dissolved in 100 L 2 (v/v) ACN in 0.1 (v/v) TFA.Liquid chromatographyPeptide separation was done by reversed phase chromatography using a nano-HPLC system (Dionex GmbH, Idstein, Germany) which consists of an autosampler (Famos), a loading pump (Switchos), a gradient pump (Ultimate) and a microfraction collector (Probot). An aliquot of 20 L of each sample was injected onto a C18 trap column (PepMap 300 m ?5 mm, 3 m, 100 ? Dionex) with 2 (v/v) acetonitrile in 0.1 (v/v) TFA at a flow rate of 30 L/min. After switching the trap column into the nanoflow (200 nL/min), the peptides were eluted onto a separation column (PepMap, C18 reversed phase material, 75 m ?150 mm, 3 m, 100 ? Dionex GmbH, Idstein, Germany) and separated at a flow rate of 200 nL/min using eluent A with 5 (v/v) acetonitrile in 0.1 (v/v) TFA and eluent B with 80 (v/v) acetonitrile in 0.1 (v/v) TFA with a gradient from 10 to 40 (v/v) eluent B in 134 min and 40 (v/v) to 100 (v/v) eluent B in 10 min. 384 fractions of 94 nL and spotting time of 23 s were spotted directly onto a manually prespotted MALDI Anchor Chip 600/384 target plate (Bruker Daltonics GmbH, Bremen, Germany) with a continuous sheath flow of 2.5 L/min 5 (v/v) acetonitrile in 0.1 (v/v) TFA.MALDI TOF/TOFcalibration 0.3 L of peptide calibration standard (Bruker Daltonik GmbH) was spotted onto the target calibration spots. Mass spectra were recorded with an Ultraflex TOF/TOF I mass spectrometer (Bruker Daltonics GmbH, Bremen, Germany) Vorinostat operating with FlexControl 2.4, FlexAnalysis 2.4, BioTools 3.0 and WARP-LC 1.1 software. To identify proteins, searches against the MSDB database (version 2007, 148 210 sequences for human) using Mascot 2.2 software (Matrix Science, inhouse server) were carried out. For FDR calculation the BTTask.tas of each run was converted to .mgf by software programm TasFileMGFExporter (provided by Bruker Daltonik GmbH), all .mgf files from one sample were combined by Notepad++ v.5.6.8, and automatic.