title: Quantitative mass spectrometric profiling of cancer-cell proteomes derived from liquid and solid tumors.
abstract: 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.
authors: Institute of Pathology, University Medical Center, Göttingen; Hanibal.bohnenberger@med.uni-goettingen.de.
authors: Hanibal
authors: H
authors: Bohnenberger
authors: Institute of Pathology, University Medical Center, Göttingen.
authors: Philipp
authors: P
authors: Ströbel
authors: Department of Hematology/Oncology, Goethe University of Frankfurt.
authors: Sebastian
authors: S
authors: Mohr
authors: Bioanalytical Mass Spectrometry Group, Max Planck Institute for Biophysical Chemistry.
authors: Jasmin
authors: J
authors: Corso
authors: Department of Hematology/Oncology, Goethe University of Frankfurt.
authors: Tobias
authors: T
authors: Berg
authors: Bioanalytical Mass Spectrometry Group, Max Planck Institute for Biophysical Chemistry; Bioanalytics, Institute of Clinical Chemistry, University Medical Center, Göttingen.
authors: Henning
authors: H
authors: Urlaub
authors: Bioanalytical Mass Spectrometry Group, Max Planck Institute for Biophysical Chemistry; Bioanalytics, Institute of Clinical Chemistry, University Medical Center, Göttingen.
authors: Christof
authors: C
authors: Lenz
authors: Department of Hematology/Oncology, Goethe University of Frankfurt; German Cancer Consortium; German Cancer Research Center.
authors: Hubert
authors: H
authors: Serve
authors: Department of Hematology/Oncology, Goethe University of Frankfurt; German Cancer Consortium; German Cancer Research Center; Thomas.Oellerich@kgu.de.
authors: Thomas
authors: T
authors: Oellerich
mesh_terms: D002853:Chromatography, Liquid / Q000379:methods; D005434:Flow Cytometry / Q000379:methods; D006801:Humans; D061346:Laser Capture Microdissection / Q000379:methods; D013058:Mass Spectrometry / Q000379:methods*; D009369:Neoplasms / Q000737:chemistry* / Q000378:metabolism*; D020543:Proteome / Q000032:analysis* / Q000378:metabolism*; D040901:Proteomics / Q000379:methods
chemical_list: D020543:Proteome