Proteomic Analysis to Identify Novel Circulating Breast Cancer Markers

Abstract

Abstract : Serum protein profiling using mass spectrometry is a promising approach to identify novel circulating breast cancer markers. In this study, serum was fractionated to deplete highly abundant proteins. After protein digestion, we used liquid chromatography mass spectrometry (LC-MS) to develop diagnostic fingerprints using bioinformatic techniques. Samples were randomized prior to fractionation and mass spectrometry testing. Each fraction was digested with trypsin and subsequently analyzed by LC-MS. Peptides were targeted based on the disease to control peak intensity ratios measured in the averages of all mass spectra in each group and t-tests of the intensity of each individual peak. A series of preprocessing steps (spectral alignment, baseline subtraction, normalization) were employed to produce an expansive list of peptides for further investigation and sequencing. The antibody columns removed 12 of the most abundant proteins in serum. Using LC-MS and bioinformatic analysis we found 17-36 differentially expressed peaks in the Cancer vs. Healthy groups. Efforts are ongoing to identify targeted peptide ion signals using tandem matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS/MS). Serum fractionation using specific antibody columns followed by LC-MS and bioinformatic analysis is a feasible approach to peptide profiling in healthy women and breast cancer patients.