One step microelectroelution concentration method for efficient coupling of sodium dodecylsulfate gel electrophoresis and matrix-assisted laser desorption time-of-flight mass spectrometry for protein analysis.
The coupling of the widely used separation technique of conventional sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) with the mass accuracy measurement capability of mass spectrometry (MS) provides a very powerful analytical technique. However, at present, there is no simple, definitive method for coupling the two methods. Typically, separated proteins are extracted from the gel, either as the native protein or as a peptide mixture after in-gel proteolytic digestion, and then analyzed by mass spectrometry. However, the various extraction techniques described previously have been labor intensive and require a large number of steps. The mass spectrometry analysis of very low concentrations of in vivo derived proteins requires minimum sample handling and on-line concentration. Therefore, we have developed an efficient microelectroelution technique that is applied in a single step manner and contains an on-line concentration device. Initial results from this system have shown a high efficiency of analyte elution from the gel and a simple, robust technique for the coupling of SDS-containing gels with MALDI-TOF-MS analysis and a capability of analyzing proteins at the subpicomole level.
Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, Minnesota, USA.
N J
NJ
Clarke
F
F
Li
A J
AJ
Tomlinson
S
S
Naylor
D000596:Amino Acids / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D008832:Microchemistry; D011506:Proteins / Q000032:analysis*; D012967:Sodium Dodecyl Sulfate; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D000596:Amino Acids; D011506:Proteins; D012967:Sodium Dodecyl Sulfate