Recovery of sodium dodecyl sulfate-proteins from gel electrophoretic bands in a single electroelution step for mass spectrometric analysis.
Mass spectrometric analysis of proteins derived from bands in gel electrophoresis is incompatible with the covalent fluorescent labeling of the protein. Thus, if one wishes to take advantage of the capacity for computer-directed electroelution of electrophoresis apparatus with intermittent fluorescent scanning of the migration path, the protein must be labeled fluorescently in a noncovalent, reversible fashion. This was recently achieved by staining of SDS-proteins with Cascade blue and electrophoresis in barbital buffer. However, the method was not a practical one for the purpose of isolating proteins from gel electrophoretic bands and their transfer into the mass spectrometer for three reasons: (i) Ten consecutive electroelution steps were required to obviate pH changes in the electroelution chamber; (ii) electroeluates from six gel electrophoretic lanes needed to be pooled; (iii) excessive protein loads ranging from 7 to 33 microg/pool were required. The present study reports the solution to those three problems. Mass spectrometric (MALDI-TOF) characterization of five proteins was demonstrated (i) after a single electroelution step; (ii) using electroelution from a single gel of 0.3-cm(2) cross-sectional area; and (iii) using a protein load of 2 (in one case 4) microg. However, the migration rates of the Cascade blue-SDS-protein-barbital complexes derived from proteins with widely varying molecular weights proved to be the same. Thus, despite the three advances made, the method to date remains restricted to samples of single proteins.
Yefimov
S
S
Section on Macromolecular Analysis, National Institutes of Health, Bethesda, Maryland 20892, USA.
Sjomeling
C
C
Yergey
A L
AL
Li
T
T
Chrambach
A
A
D004587:Electrophoresis, Agar Gel / Q000379:methods*; D005453:Fluorescence; D006863:Hydrogen-Ion Concentration; D011506:Proteins / Q000737:chemistry / Q000302:isolation & purification*; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry / Q000302:isolation & purification*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D011506:Proteins; D012967:Sodium Dodecyl Sulfate