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.
Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, 01307 Dresden, Germany.
Andrej
A
Vasilj
Marc
M
Gentzel
Elke
E
Ueberham
Rolf
R
Gebhardt
Andrej
A
Shevchenko
D025601:Adenomatous Polyposis Coli Protein / Q000172:deficiency / Q000235:genetics; D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000592:standards; D004591:Electrophoresis, Polyacrylamide Gel; D057927:Hydrophobic and Hydrophilic Interactions; D008099:Liver / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D020411:Oligonucleotide Array Sequence Analysis; D010449:Peptide Mapping; D020543:Proteome / Q000302:isolation & purification / Q000378:metabolism*; D040901:Proteomics; D012015:Reference Standards; D012984:Software; D053719:Tandem Mass Spectrometry / Q000592:standards; D046888:Tissue Array Analysis; D057825:Two-Dimensional Difference Gel Electrophoresis
D025601:Adenomatous Polyposis Coli Protein; D020543:Proteome