Sequential electroelution and mass spectroscopic identification of intact sodium dodecyl sulfate-proteins labeled with 5(6)-carboxyfluorescein-N-hydroxysuccinimide ester.
A gel electrophoresis apparatus capable of scanning the migration path fluorometrically and of computer-directed electroelution of bands was applied to the mass spectrometric identification of sequentially electroeluted 5(6)-carboxyfluorescein-N-hydrosuccinimide ester (FLUOS)-labeled sodium dodedyl sulfate (SDS)-proteins. The masses of four electroeluted SDS-proteins under study determined by matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) spectrometry are changed by 1% due to their reaction with FLUOS in a 1:5 molar ratio of protein:label, allowing for the identification of the labeled intact proteins on the basis of mass. More importantly, the partial (10 or 50%) derivatization of proteins with FLUOS does not preclude their tryptic hydrolysis, and identification of the protein on the basis of the mass spectrometric analysis of its tryptic peptides. Potentially, the procedure allows for the automated mass spectrometric identification of SDS-proteins globally labeled with FLUOS and electrophoretically separated, without need for any gel sectioning.
Yefimov
S
S
Laboratory of Cellular and Molecular Biophysics, NICHD, National Institutes of Health, Bethesda, MD, USA.
Yergey
A L
AL
Chrambach
A
A
D000818:Animals; D002256:Carbonic Anhydrases / Q000032:analysis / Q000302:isolation & purification; D002417:Cattle; D016287:Computer Peripherals; D003902:Detergents; D004566:Electrodes; D004587:Electrophoresis, Agar Gel; D004591:Electrophoresis, Polyacrylamide Gel / Q000295:instrumentation / Q000379:methods*; D005452:Fluoresceins / Q000032:analysis*; D005456:Fluorescent Dyes / Q000032:analysis*; D005470:Fluorometry / Q000379:methods*; D006868:Hydrolysis; D010047:Ovalbumin / Q000032:analysis / Q000302:isolation & purification; D010940:Plant Proteins / Q000032:analysis / Q000302:isolation & purification; D011506:Proteins / Q000032:analysis / Q000302:isolation & purification*; D012710:Serum Albumin, Bovine / Q000032:analysis / Q000302:isolation & purification; D012967:Sodium Dodecyl Sulfate; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013194:Staining and Labeling / Q000379:methods; D014357:Trypsin / Q000494:pharmacology; D014360:Trypsin Inhibitor, Kunitz Soybean / Q000032:analysis / Q000302:isolation & purification
D003902:Detergents; D005452:Fluoresceins; D005456:Fluorescent Dyes; D010940:Plant Proteins; D011506:Proteins; C097499:fluorescein carboxylate; D012710:Serum Albumin, Bovine; D012967:Sodium Dodecyl Sulfate; D010047:Ovalbumin; D014360:Trypsin Inhibitor, Kunitz Soybean; D014357:Trypsin; D002256:Carbonic Anhydrases