In-depth analyses of
cancer cell
proteomes are needed to elucidate oncogenic pathomechanisms, as well as to identify potential drug targets and diagnostic
biomarkers. However, methods for quantitative proteomic characterization of patient-derived
tumors and in particular their cellular subpopulations are largely lacking. Here we describe an experimental set-up that allows quantitative analysis of
proteomes of
cancer cell subpopulations derived from either liquid or solid
tumors. This is achieved by combining cellular enrichment strategies with quantitative Super-SILAC-based mass spectrometry followed by bioinformatic data analysis. To enrich specific cellular subsets, liquid
tumors are first immunophenotyped by flow cytometry followed by FACS-sorting; for solid
tumors,
laser-capture microdissection is used to purify specific cellular subpopulations. In a second step,
proteins are extracted from the purified cells and subsequently combined with a
tumor-specific, SILAC-labeled spike-in standard that enables
protein quantification. The resulting
protein mixture is subjected to either gel electrophoresis or Filter Aided Sample Preparation (FASP) followed by tryptic digestion. Finally, tryptic
peptides are analyzed using a hybrid quadrupole-orbitrap mass spectrometer, and the data obtained are processed with bioinformatic software suites including MaxQuant. By means of the workflow presented here, up to 8,000
proteins can be identified and quantified in patient-derived samples, and the resulting
protein expression profiles can be compared among patients to identify diagnostic proteomic signatures or potential drug targets.