Proteinpilot descriptive statistics template pdst

Protein concentrations were measured by BCA-protein assay. Approximately 30 μg of proteins were separated by SDS-PAGE using 10% Bis-Tris Nupage gels. Serial gel slices were excised and diced into smaller fragments. Samples were reduced with 10 mM dithiothreitol in 25 mM NH4HCO3 at 56 °C for 1 h and alkylated with 55 mM iodoacetamide for 45 min at RT. In-gel trypsin digestion was performed using 10 ng/μl of sequencing grade modified porcine trypsin (Promega) diluted in 505 mM NH4HCO3 at 37 °C overnight. Peptides were extracted with 0.5% formic acid and 50% acetonitrile. Following evaporation of acetonitrile, peptides were purified using a ZipTipC18 column (Millipore). The volume of each eluted sample was reduced in a Speedvac to 5 μl to evaporate acetonitrile and adjusted to 20 μl with 0.1% formic acid prior to LC–MS/MS analysis. An AB SCIEX TripleTOF 5600 System (Foster City) equipped with an Eksigent nanoLC Ultra and ChiPLC-nanoflex (Eksigent) in Trap Elute configuration was employed for LC-MS measurement. The acquired mass spectrometric raw data was processed using ProteinPilot 5.0 software (AB SCIEX) with the Paragon search mode. The ProteinPilot Descriptive Statistics Template (PDST, AB SCIEX) was used for alignment of multiple results and evaluation of the false discovery rate.