Electronic gel protein transfer and identification using matrix-assisted laser desorption/ionization-mass spectrometry.
An electronic protein transfer technique is described for achieving the rapid and efficient recovery of sodium dodecyl sulfate (SDS)-protein complexes from polyacrylamide gels. This process involves the use of small-dimension capillaries in physical contact with a resolved protein band within the polyacrylamide gel, providing a large potential drop and high electric field strength at the capillary/gel interface. Several factors controlling the electronic protein transfer, including the applied electric field strength, the electrophoresis buffer concentration, and the capillary dimension, are studied to further enhance the use of field-amplification for sample stacking of extracted SDS-protein complexes. As a result of sample stacking, the extracted proteins from a 50 ng gel loading are present in a narrow ( approximately 80 nL) and highly concentrated (0.46 mg/mL or 3.3 x 10(-5) M for cytochrome c) solution plug. Three model proteins with molecular mass ranging from 14 kDa (cytochrome c) to 116 kDa (beta-galactosidase) are stained by Coomassie blue and electrophoretically extracted from gels with protein loadings as low as 50 ng. The capillary format of the electronic protein transfer technique allows direct deposition of extracted proteins onto a matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) target. Various matrices and solvent compositions are evaluated for the analysis of extracted and concentrated SDS-protein complexes using MALDI-MS. The electronic protein transfer technique, when operated under optimized conditions, is demonstrated for the effective (>70% recovery), speedy (less than 5 min), and sensitive MS identification of gel resolved proteins (as low as 50 ng).
Cooper
Jonathan W
JW
Calibrant Biosystems, Rockville, MD, USA.
Gao
Jun
J
Lee
Cheng S
CS
D045304:Cytochromes c / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D005782:Gels / Q000737:chemistry; D011506:Proteins / Q000737:chemistry; D012394:Rosaniline Dyes / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D001616:beta-Galactosidase / Q000737:chemistry*
D005782:Gels; D011506:Proteins; D012394:Rosaniline Dyes; C048139:Coomassie blue; D045304:Cytochromes c; D001616:beta-Galactosidase