Quantitative proteomic analysis of fresh-frozen human breast tissue and tumor samples using microLC-SWATH-MS

Future improvements in the field of breast cancer prevention, diagnosis and treatment require deeper knowledge and better understanding of the molecular processes and signaling pathways that lead to the transformation of normal cells to cancer ones and the disease progression. Quantitative proteome profiling of fresh frozen breast tissue and tumor samples may accelerate and facilitate this process. Liquid chromatography (LC) hyphenated to tandem mass spectrometry (MS/MS) technique in data-independent SWATH acquisition mode is a powerful tool for such profiling, because all the precursor ions are fragmented there, which enables label-free identification and quantification of theoretically all proteins in the sample. In this study we performed microLC separation before SWATH-MS analysis. The spectral library, which is necessary for SWATH quantification of the investigated samples, consisted of proteins identified by protein database search of the MS/MS spectra obtained by the information-dependent acquisition (IDA) screening of both normal human breast tissue and breast tumor samples, which were additionally enriched in low abundant proteins by immunoaffinity depletion from 14 high abundant serum proteins. Using the described methodology we detected 547 proteins in all qualitative experiments, which served then as a spectral library for further label-free SWATH quantification of separate breast tumor and corresponding normal breast tissue samples of 8 patients. Among them, 299 proteins were successfully quantified. Levels of 188 of them varied significantly (p<0.05) between normal breast tissue and breast tumor samples. 31proteins were up-regulated and 14 were down-regulated at least two fold in breast tumors comparing to normal breast tissues.