Pre-extraction sample handling by automated frozen disruption significantly improves subsequent proteomic analyses.
Here we quantitatively characterize two common homogenization strategies in the analysis of tissue proteomes: classical manual homogenization (MH) and an automated frozen disruption (AFD) technique. In a variety of tissues, many proteins were more efficiently extracted, resolved and detected, with high reproducibility after AFD, amounting to as much as 2% of the total resolved proteome. The benefits of AFD over MH are 2-fold: (1) AFD yields a much more thorough homogenate than MH; and (2) as a deep frozen alternative, AFD maintains a level of biological complexity that is not retained during MH. Thus, AFD coupled with refined 2DE protocols and Sypro Ruby staining yields quantitative proteomic analyses.
Butt
R Hussain
RH
Department of Physiology & Biophysics, Faculty of Medicine, University of Calgary, Calgary, AB, Canada.
Coorssen
Jens R
JR
D000818:Animals; D001921:Brain; D045424:Complex Mixtures / Q000032:analysis; D015925:Cryopreservation; D015180:Electrophoresis, Gel, Two-Dimensional; D006135:Gryllidae / Q000378:metabolism*; D008110:Liver Extracts / Q000032:analysis; D051379:Mice; D009206:Myocardium / Q000378:metabolism; D010936:Plant Extracts / Q000032:analysis; D020543:Proteome; D018724:Spinacia oleracea / Q000378:metabolism*
D045424:Complex Mixtures; D008110:Liver Extracts; D010936:Plant Extracts; D020543:Proteome