Shotgun proteomics is based on the separation of peptides, generated mainly from trytic digests of complex protein mixtures, by nano-flow liquid chromatography, and subsequent identification and quantification by mass spectrometry. While gel-based proteomics may be better to quantify proteins and detect subtle cell responses to challenges in vitro, shotgun analysis is more sensitive with respect to protein identification.
In more detail, peptides are separated by nano-flow liquid chromatography (1100 Series LC system, Agilent, Palo Alto, CA) using the HPLC-Chip technology (Agilent) equipped with a 40 nl Zorbax 300SB-C18 trapping column and a 75 µm x 150 mm Zorbax 300SB-C18 separation column at a flow rate of 400 nl/min using a gradient from 0,2% formic acid and 3% ACN to 0,2% formic acid and 45% ACN over 80 minutes. Peptide identification is accomplished by MS/MS fragmentation analysis with an iontrap mass spectrometer (XCT-Ultra, Agilent) equipped with an orthogonal nanospray ion source.
The MS/MS data are interpreted by the Spectrum Mill MS Proteomics Workbench software (Version A.03.02, Agilent) and searched against the SwissProt Database (currently version 20061207) allowing for precursor mass deviation of 1,5 Da, a product mass tolerance of 0,7 Da and a minimum matched peak intensity (%SPI) of 70%. Due to previous chemical modification, carbamidomethylation of cysteines is set as fixed modification. Peptides are identified with the indicated scores, which are essentially calculated from sequence tag lengths, but also consider mass deviations. To assess the reliability of the peptide scores, we performed searches against the corresponding reverse database. 5,4% positive hits were found with peptides scoring >9,0, while 0,18% positive hits were found with peptides scoring >13,0. Consequently, we set the threshold for protein identification to at least one peptide scoring higher than 13,0. Peptide scores and sequence coverages of all identified proteins are provided in Table S2. Mass data were organized by a home-made SQL-database (programmed by Helge Wimmer).
