Quantitative mass spectrometric profiling of cancer-cell proteomes derived from liquid and solid tumors

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 character
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.
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Metadaten
Author:Hanibal Bohnenberger, Philipp Ströbel, Sebastian Mohr, Jasmin Corso, Tobias Berg, Henning Urlaub, Christof Lenz, Hubert Serve, Thomas Oellerich
URN:urn:nbn:de:hebis:30:3-372443
DOI:http://dx.doi.org/10.3791/52435
ISSN:1940-087X
Pubmed Id:http://www.ncbi.nlm.nih.gov/pubmed?term=25867170
Parent Title (English):Journal of visualized experiments : JoVE
Document Type:Article
Language:English
Date of Publication (online):2015/02/27
Date of first Publication:2015/02/27
Publishing Institution:Universitätsbibliothek Johann Christian Senckenberg
Release Date:2015/06/01
Tag:Issue 96; Medicine; Proteomics; formalin-fixed and paraffin-embedded tissue (FFPE); laser-capture microdissection; leukemia; quantitative mass spectrometry; solid tumors; spike-in SILAC
Volume:96
Issue:e52435
Pagenumber:8
Note:
Copyright © 2015, Journal of Visualized Experiments This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial License, which permits non-commercial use, distribution, and reproduction, provided the original work is properly cited.
HeBIS PPN:369178726
Institutes:Medizin
Dewey Decimal Classification:610 Medizin und Gesundheit
Sammlungen:Universitätspublikationen
Licence (English):License LogoCreative Commons - Namensnennung-Nicht kommerziell 4.0

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