Breast cancer tumors
The project analyzed 88 breast cancer clinical samples, including lymph node negative and positive primary tumors, lymph node metastases, and healthy tissue as control. All samples were combined with a super-SILAC mix that served as an internal standard for quantification.
Sample Processing Protocol
Formalin-fixed paraffin-embedded tissue were extracted and trypsin digested following the FFPE-FASP protocol. Some of the samples were further fractionated using strong anion exchange in a stageTip format. Peptides were separated on C18 50 cm PepMap columns using the EASY nLC1000 UHPLC connected to the Q-Exactive mass spectrometer through the EASY-spray ionization source.
Data Processing Protocol
Raw files were analyzed with MaxQuant version 22.214.171.124. The Andromeda search engine was used for database search against the Human Uniprot database. Variable modifications were methionine oxidation and N-terminal acetylation. Fixed modification was carbamidomethyl cysteine. The forward/decoy approach was utilized to determine 1% FDR on the peptide and protein levels.
Pozniak Y, Balint-Lahat N, Rudolph JD, Lindskog C, Katzir R, Avivi C, Pontén F, Ruppin E, Barshack I, Geiger T. System-wide Clinical Proteomics of Breast Cancer Reveals Global Remodeling of Tissue Homeostasis. Cell Syst. 2016 Mar 23;2(3):172-184 PubMed: 27135363