Tissue Proteomics by One-Dimensional Gel Electrophoresis Combined with Label-Free Protein Quantification

Label-free methods streamline quantitative proteomics of tissues by alleviating the need for metabolic labeling of proteins with stable isotopes. Here we detail and implement solutions to common problems in label-free data processing geared toward tissue proteomics by one-dimensional gel electrophor...

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Bibliographic Details
Published in:Journal of proteome research 2012-07, Vol.11 (7), p.3680-3689
Main Authors: Vasilj, Andrej, Gentzel, Marc, Ueberham, Elke, Gebhardt, Rolf, Shevchenko, Andrej
Format: Article
Language:English
Subjects:
Adenomatous Polyposis Coli Protein - deficiency
Adenomatous Polyposis Coli Protein - genetics
Animals
Chromatography, High Pressure Liquid - standards
Electrophoresis, Polyacrylamide Gel
Hydrophobic and Hydrophilic Interactions
Liver - metabolism
Mice
Mice, Inbred C57BL
Mice, Knockout
Oligonucleotide Array Sequence Analysis
Peptide Mapping
Proteome - isolation & purification
Proteome - metabolism
Proteomics
Reference Standards
Software
Tandem Mass Spectrometry - standards
Tissue Array Analysis
Two-Dimensional Difference Gel Electrophoresis
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Summary:Label-free methods streamline quantitative proteomics of tissues by alleviating the need for metabolic labeling of proteins with stable isotopes. Here we detail and implement solutions to common problems in label-free data processing geared toward tissue proteomics by one-dimensional gel electrophoresis followed by liquid chromatography tandem mass spectrometry (geLC MS/MS). Our quantification pipeline showed high levels of performance in terms of duplicate reproducibility, linear dynamic range, and number of proteins identified and quantified. When applied to the liver of an adenomatous polyposis coli (APC) knockout mouse, we demonstrated an 8-fold increase in the number of statistically significant changing proteins compared to alternative approaches, including many more previously unidentified hydrophobic proteins. Better proteome coverage and quantification accuracy revealed molecular details of the perturbed energy metabolism.
ISSN:1535-3893
1535-3907
DOI:10.1021/pr300147z