Comparison of label-free and label-based strategies for proteome analysis of hepatoma cell lines.

Abstract

Within the past decade numerous methods for quantitative proteome analysis have been developed of which all exhibit particular advantages and disadvantages. Here, we present the results of a study aiming for a comprehensive comparison of ion-intensity based label-free proteomics and two label-based approaches using isobaric tags incorporated at the peptide and protein levels, respectively. As model system for our quantitative analysis we used the three hepatoma cell lines HepG2, Hep3B and SK-Hep-1. Four biological replicates of each cell line were quantitatively analyzed using an RPLC-MS/MS setup. Each quantification experiment was performed twice to determine technical variances of the different quantification techniques. We were able to show that the label-free approach by far outperforms both TMT methods regarding proteome coverage, as up to threefold more proteins were reproducibly identified in replicate measurements. Furthermore, we could demonstrate that all three methods show comparable reproducibility concerning protein quantification, but slightly differ in terms of accuracy. Here, label-free was found to be less accurate than both TMT approaches. It was also observed that the introduction of TMT labels at the protein level reduces the effect of underestimation of protein ratios, which is commonly monitored in case of TMT peptide labeling. Previously reported differences in protein expression between the particular cell lines were furthermore reproduced, which confirms the applicability of each investigated quantification method to study proteomic differences in such biological systems. This article is part of a Special Issue entitled: Biomarkers: A Proteomic Challenge.

• Authors: Dominik A Megger, Leona L Pott, Maike Ahrens, Juliet Padden, Thilo Bracht, Katja Kuhlmann, Martin Eisenacher, Helmut E Meyer, Barbara Sitek
• Journal: Biochimica et biophysica acta (Biochim Biophys Acta) Vol. 1844 Issue 5 Pg. 967-76 (May 2014) ISSN: 0006-3002 [Print] Netherlands
• PMID: 23954498 (Publication Type: Comparative Study, Journal Article, Research Support, Non-U.S. Gov't)
• Copyright: © 2013 Elsevier B.V. All rights reserved.

Chemical References

• Biomarkers, Tumor
• Neoplasm Proteins
• Peptide Fragments
• Proteome

Topics

• Biomarkers, Tumor (analysis)
• Carcinoma, Hepatocellular (diagnosis, metabolism)
• Chromatography, Liquid
• Humans
• Isotope Labeling (methods)
• Liver Neoplasms (diagnosis, metabolism)
• Neoplasm Proteins (analysis)
• Peptide Fragments (analysis)
• Proteome (analysis)
• Proteomics (methods)
• Staining and Labeling
• Tandem Mass Spectrometry
• Tumor Cells, Cultured