Spectrum mill ms proteomics workbench software

Silver-stained protein bands were retrieved from SDS-PAGE gels. Proteins were in-gel digested with trypsin in 25 mM ammonium bicarbonate overnight at 37 °C, followed by extraction of peptides with 60% acetonitrile in 0.1% formic acid. Samples were dried in a Savant SpeedVac concentrator (Thermo Fisher Scientific), and then dissolved in 3% acetonitrile in 0.1% formic acid. The peptides were separated on a 43-mm HPLC C18 ProtID-Chip (Agilent Technologies), followed by data-dependent auto-MS/MS on an nano-electrospray-ionization 6520 Q-TOF tandem MS spectrometer (Agilent Technologies). Protein identifications were achieved by searching Swiss-Prot Homo sapiens protein database using Spectrum Mill MS Proteomics Workbench software (version B.04.00; Agilent Technologies) with a global false discovery rate of 1%.

MS/MS data were interpreted by the Spectrum Mill MS Proteomics Workbench software (Version A.03.03, Agilent) and searched against the SwissProt database for human proteins (version 14.3 containing 20,328 entries) allowing for a precursor mass deviation of 1.5 Da, a product mass tolerance of 0.7 Da, a minimum matched peak intensity (%SPI) of 70% and carbamidomethylation of cysteines was set as fixed modification. For further modifications only oxidized methionine was considered. The listed peptides were identified with scores calculated from sequence tag lengths and mass deviations.