Transfer of SDS-proteins from gel electrophoretic zones into mass spectrometry, using electroelution of the band into buffer without sectioning of the gel.
Five SDS-proteins, ranging in molecular weight from 14 to 66 kDa, were detected without covalent fluorescent labeling by the automated gel electrophoresis apparatus with intermittent fluorescence scanning (HPGE apparatus, LabIntelligence) during electrophoresis in barbiturate buffer in the presence of Cascade Blue. The SDS-proteins were electroeluted from the gel into 220 microl of buffer by a modification of the procedure of Gombocz and Cortez. The electroeluate was freed of SDS, ultrafiltered and subjected to MALDI-TOF mass spectrometry. The masses of the five native proteins were found to be maintained after electrophoresis and electroelution in the presence of the potential contaminants SDS, barbituric acid and Cascade Blue. The procedure of protein transfer from SDS-PAGE into mass spectrometry, without excision of bands, gel maceration and protein recovery by diffusion, therefore is shown to be suitable for the identification by mass of intact proteins derived from gel electrophoretic bands.
Yefimov
S
S
Section on Macromolecular Analysis, Laboratory of Cellular and Molecular Biophysics, NICHD, National Institutes of Health, Bethesda, MD 20892-1580, USA.
Yergey
A L
AL
Chrambac
A
A
D001671:Biochemistry / Q000379:methods*; D002021:Buffers; D002256:Carbonic Anhydrases / Q000032:analysis / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D005456:Fluorescent Dyes / Q000032:analysis / Q000737:chemistry; D007768:Lactalbumin / Q000032:analysis / Q000737:chemistry; D009942:Organometallic Compounds / Q000032:analysis / Q000737:chemistry; D009943:Organophosphorus Compounds / Q000032:analysis / Q000737:chemistry; D010047:Ovalbumin / Q000032:analysis / Q000737:chemistry; D011506:Proteins / Q000032:analysis* / Q000737:chemistry*; D012710:Serum Albumin, Bovine / Q000032:analysis / Q000737:chemistry; D012967:Sodium Dodecyl Sulfate / Q000302:isolation & purification; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D014360:Trypsin Inhibitor, Kunitz Soybean / Q000032:analysis / Q000737:chemistry
D002021:Buffers; D005456:Fluorescent Dyes; D009942:Organometallic Compounds; D009943:Organophosphorus Compounds; D011506:Proteins; C066850:Cascade Blue; D012710:Serum Albumin, Bovine; D012967:Sodium Dodecyl Sulfate; D010047:Ovalbumin; D007768:Lactalbumin; D014360:Trypsin Inhibitor, Kunitz Soybean; D002256:Carbonic Anhydrases