Peptide mapping and microsequencing of proteins separated by SDS-PAGE after limited in situ acid hydrolysis.
A method is described for the isolation of peptide fragments from proteins separated by polyacrylamide gel electrophoresis. After completion of the electrophoresis step, gels are stained with Ponceau S or Coomassie Blue. Gel portions containing protein stained with Ponceau S are excised and transferred to borosilicate glass digestion tubes containing 0.9 ml of 1 mM NaOH or 5 mM Na2HPO4. After complete dissociation of the dye from the protein, 0.1 ml of 20% formic acid is added and the protein is hydrolyzed in situ at 112 degrees C for four hours. Subsequently the acid solution is made 10% in acetonitrile and chromatographed as such on a C18 (C4) reversed-phase column using an appropriate large-volume sample loading syringe and injection loop. Proteins stained with Coomassie Blue can be hydrolyzed in situ after complete removal of the dye with an aqueous solution containing 40% acetone, 10% triethylamine and 5% acetic acid. The gel slices are next washed with HPLC-grade water and protein is hydrolyzed in 2% formic acid under standard conditions. Gel-related contaminants do not interfere with the peptide separation under the proper conditions of HPLC analysis.
Vanfleteren
J R
JR
Laboratory of Animal Morphology and Systematics, University of Ghent, Belgium.
Raymackers
J G
JG
Van Bun
S M
SM
Meheus
L A
LA
D000595:Amino Acid Sequence; D001059:Apoproteins / Q000737:chemistry; D001391:Azo Compounds; D002256:Carbonic Anhydrases / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid; D003574:Cytochrome c Group / Q000737:chemistry; D045304:Cytochromes c; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D006868:Hydrolysis; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry*; D010449:Peptide Mapping / Q000379:methods*; D011506:Proteins / Q000302:isolation & purification; D012394:Rosaniline Dyes; D014168:Transferrin / Q000737:chemistry
D001059:Apoproteins; D001391:Azo Compounds; D003574:Cytochrome c Group; D010446:Peptide Fragments; D011506:Proteins; D012394:Rosaniline Dyes; D014168:Transferrin; C048139:Coomassie blue; D045304:Cytochromes c; D002256:Carbonic Anhydrases; C032756:ponceau S