An effective skeletal muscle prefractionation method to remove abundant structural proteins for optimized two-dimensional gel electrophoresis.
Proteomic analysis of biological samples in disease models or therapeutic intervention studies requires the ability to detect and identify biologically relevant proteins present in relatively low concentrations. The detection and analysis of these low-level proteins is hindered by the presence of a few proteins that are expressed in relatively high concentrations. In the case of muscle tissue, highly abundant structural proteins, such as actin, myosin, and tropomyosin, compromise the detection and analysis of more biologically relevant proteins. We have developed a practical protocol which exploits high-pH extraction to reduce or remove abundant structural proteins from skeletal muscle crude membrane preparations in a manner suitable for two dimensional gel electrophoresis. An initial whole-cell muscle lysate is generated by homogenization of powdered tissue in Tris-base. This lysate is subsequently partitioned into a supernatant and pellet containing the majority of structural proteins. Treatment of the pellet with high-pH conditions effectively releases structural proteins from membrane compartments which are then removed through ultracentrifugation. Mass spectrometric identification shows that the majority of protein spots reduced or removed by high-pH treatment were contractile proteins or contractile-related proteins. Removal of these proteins enabled successful detection and identification of minor proteins. Structural protein removal also results in significant improvement of gel quality and the ability to load higher amounts of total protein for the detection of lower abundant protein classes.
Jarrold
Bradley
B
Procter & Gamble Pharmaceuticals, Inc., Health Care Research Center, Mason, OH 45040, USA.
DeMuth
Jeffrey
J
Greis
Kenneth
K
Burt
Thomas
T
Wang
Feng
F
D000818:Animals; D002458:Cell Fractionation; D005591:Chemical Fractionation; D003285:Contractile Proteins / Q000302:isolation & purification; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D006863:Hydrogen-Ion Concentration; D009124:Muscle Proteins / Q000302:isolation & purification*; D018482:Muscle, Skeletal / Q000737:chemistry*; D040901:Proteomics / Q000379:methods; D051381:Rats
D003285:Contractile Proteins; D009124:Muscle Proteins