Human triple negative breast cancer tissues

Tissue samples were cut into 8-µm frozen sections and then microdissected to obtain ~4,000 breast cancer epithelial cells using a P.A.L.M. MicroBeam system (Carl Zeiss MicroImaging, GmbH, Munich, Germany). Proteins were extracted from microdissected cells, followed by denaturation, reduction and alkylation. Protein samples were digested at 37oC for 4 h using MS-grade trypsin (Promega, Madison, WI, USA) at a 1:4 (enzyme:protein) ratio, and then acidified for further analysis. Global proteomic profiles of 126 TNBC samples were recorded on a nanoscale LC hyphenated LTQ-Orbitrap-XL MS system (ThermoElectron, Bremen, Germany). Peptide mixtures were separated on the nLC system (Ultimate 3000, Dionex, Amsterdam, The Netherlands) with a 3-h binary gradient (mobile phase A: H2O, mobile phase B: ACN) in a 3-μm C18 silica packed 50-cm capillary column with 75-μm inner-diameter. Mass spectra were acquired over a mass-to-charge ratio (m/z) range of 400–1,800 at a resolving power of 30,000 at 400 m/z. The top five most intensive parent ions from the full scan were isolated and fragmented by collisional activated dissociation in the linear ion trap. Dynamic exclusion was used to increase number of parent ions selected for fragmentation. MaxQuant search used Carbamidomethylation as a fixed modification for cysteine-containing peptides, and oxidation of methionine and N-terminal acetylation as variable modification.