TY  - JOUR
A1  - Bohnenberger, Hanibal
A1  - Ströbel, Philipp
A1  - Mohr, Sebastian
A1  - Corso, Jasmin
A1  - Berg, Tobias
A1  - Urlaub, Henning
A1  - Lenz, Christof
A1  - Serve, Hubert
A1  - Oellerich, Thomas
T1  - Quantitative mass spectrometric profiling of cancer-cell proteomes derived from liquid and solid tumors
T2  - Journal of visualized experiments : JoVE
N2  - 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.
KW  - Medicine
KW  - Issue 96
KW  - Proteomics
KW  - solid tumors
KW  - leukemia
KW  - formalin-fixed and paraffin-embedded tissue (FFPE)
KW  - laser-capture microdissection
KW  - spike-in SILAC
KW  - quantitative mass spectrometry
Y1  - 2015
UR  - http://publikationen.ub.uni-frankfurt.de/frontdoor/index/index/docId/37244
UR  - https://nbn-resolving.org/urn:nbn:de:hebis:30:3-372443
SN  - 1940-087X
N1  - 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.
VL  - 96
IS  - e52435
ER  -