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 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.