As more and more alternative treatments become available for breast carcinoma, there is a need to stratify patients and individual molecular information seems to be suitable for this purpose. In this study, we applied label-free protein quantitation by nanoscale LC-MS and investigated whether this approach could be used for defining a proteome signature for invasive ductal breast carcinoma. Tissue samples from healthy breast and tumor were collected from three patients. Protein identifications were based on LC-MS peptide fragmentation data which were obtained simultaneously to the quantitative information. Hereby, an invasive ductal breast carcinoma proteome signature was generated which contains 60 protein entries. The on-column concentrations for osteoinductive factor, vimentin, GAP-DH, and NDKA are provided as examples. These proteins represent distinctive gene ontology groups of differentially expressed proteins and are discussed as risk markers for primary tumor pathogenesis. The developed methodology has been found well applicable in a clinical environment in which standard operating procedures can be kept; a prerequisite for the definition of molecular parameter sets that shall be capable for stratification of patients.