Human breast cancer cell line LC-MS/MS
Human breast cancer cell lines were pooled according to ER/Her2 status. The whole cell lysate samples were reduced and treated with iodoacetamide and then digested with trypsin. The samples were separated into 6 fractions by online high pH reverse-phase fractionation and then analyzed by reverse-phase chromatography coupled to a Thermo LTQ-Orbitrap Velos mass spectrometer. MS/MS files were searched against version 3.69 of the Human International Protein Index (IPI) sequence database with decoy sequences by the X!Tandem database search engine with k-score plugin with the following parameters: tryptic enzyme constraint allowing for up to two missed cleavages. Peptide MH+ mass tolerances set at 2.0 Da with post search filtering of precursor mass to 50 ppm. Oxidized methionine as a variable modification and carbamidomethylated cysteine as a static modification. FDR < 0.005 based on decoy search.
Sample Processing Protocol
Data Processing Protocol
Jacob Kennedy, Clinical Research Division