Fast proteomic protocol for biomarker fingerprinting in cancerous cells.

The advance of novel technologies that will enable the detection of large sets of biomarker proteins, to greatly improve the sensitivity and specificity of an assay, represents a major objective in biomedical research. To demonstrate the power of mass spectrometry (MS) detection for large-scale biomarker screening in cancer research, a simple, one-step approach for fast biomarker fingerprinting in complex cellular extracts is described. MCF-7 breast cancer cells were used as a model system. Fast proteomic profiling of whole cellular extracts was achieved on a linear trap quadrupole (LTQ) mass spectrometer by one of the following techniques: (a) data-dependent liquid chromatography (LC)-MS/MS of un-labeled cell extracts, (b) data-dependent LC-MS/MS with pulsed Q dissociation (PQD) detection of iTRAQ labeled samples, and (c) multiple reaction monitoring (MRM)-MS of low abundant proteins that could not be detected with data-dependent MS/MS. The data-dependent LC-MS/MS analysis of MCF-7 cells enabled the identification of 796 proteins (p<0.001) and the simultaneous detection of 156 previously reported putative cancer biomarkers. PQD detection of iTRAQ labeled cells resulted in the detection of 389 proteins and 64 putative biomarkers. MRM-MS analysis enabled the successful monitoring of a panel of low-abundance proteins in one single experiment, highlighting the utility of this technique for targeted analysis in cancer investigations. These results demonstrate that MS-based technologies relying on a one-step separation protocol have the potential to revolutionize biomarker research and screening applications by enabling fast, sensitive and reliable detection of large panels of putative biomarkers. To further stimulate the exploration of proteins that have been previously reported in the literature to be differentially expressed in a variety of cancers, an extensive list of approximately 1100 candidate biomarkers has been compiled and included in the manuscript. Copyright 2010 Elsevier B.V. All rights reserved.