Enhanced in situ gel digestion of electrophoretically separated proteins with automated peptide elution onto mini reversed-phase columns.
An improved method for the generation and automated isolation of internal peptides by in situ gel digestion of electrophoretically separated proteins is described. To enhance the sensitivity of the method, and to reduce the amount of sample handling steps, we have automated the extraction procedure of peptides after protein cleavage in a sodium dodecyl sulfate (SDS) gel matrix. The excised protein-containing polyacrylamide bands or spots are first minced to defined particles of about 30 microns. After in situ gel digestion, the gel slurry is transferred into a mini reversed-phase column-funnel assembly in the sample loading station of the Hewlett-Packard protein sequencer. Applying nitrogen pressure elutes peptides from the gel slurry onto the reversed-phase material. The mini reversed-phase column is then placed in an in-line column adapter and connected to a micropreparative high performance liquid chromatography (HPLC) column, where separation of the peptides under standard conditions is achieved. In the work described here complete digestions and excellent peptide recoveries allowed the generation of extensive internal sequence information from low picomole amounts of proteins. The method has been routinely applied in both laboratories for two years.
Eckerskorn
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C
Max-Planck-Institute of Biochemistry, Protein Chemistry Group, Martinsried, Germany.
Grimm
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R
D000180:Acrylic Resins; D001331:Automation; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D010450:Endopeptidases / Q000378:metabolism; D005782:Gels; D013058:Mass Spectrometry / Q000379:methods*; D008666:Metalloendopeptidases / Q000378:metabolism; D011506:Proteins / Q000032:analysis*; D015203:Reproducibility of Results; D014357:Trypsin / Q000378:metabolism
D000180:Acrylic Resins; D005782:Gels; D011506:Proteins; C016680:polyacrylamide gels; D010450:Endopeptidases; D014357:Trypsin; D008666:Metalloendopeptidases; C039830:peptidyl-Lys metalloendopeptidase; C058921:endoproteinase Asp-N