Isolation and sequence analysis of proteins from mouse forebrain using two-dimensional gel electrophoresis coupled to high-pressure liquid extrusion.
This report presents a technique for recovery of mouse forebrain proteins from two-dimensional sodium dodecyl sulfate-polyacrylamide gels for subsequent primary structure determination. Proteins were visualized by Coomassie staining or salt precipitation and manually cut out of the gel. Excised spots were minced and loaded into an empty precolumn of a reversed-phase high-performance liquid chromatography system. Purified protein was extruded from a gel matrix by pressurized liquid, then separated from gel contaminants by reversed-phase gradient elution, and finally collected in siliconized tubes or on polybrene-coated filter disks for gas-phase sequencing. Several mouse and rat forebrain proteins were purified by this method and sequenced. Three previously unidentified mouse brain proteins with molecular weights of 4,000, 12,000, and 18,500 were partially sequenced and three hemoglobin fragments were structurally identified and mapped. Ribonuclease A, myoglobin, adrenocorticotropin, and bovine somatotropin were also subjected to two-dimensional (2-D) analysis and partially sequenced. Recovery values of 27-95% were obtained for extruded 14C-labeled ribonuclease, carbonic anhydrase, and bovine serum albumin out of sodium dodecyl sulfate-polyacrylamide gel electrophoretic gels. Losses resulting from the multiple handling steps of a 2-D gel separation process were also investigated. Recoveries of 12-17%, as determined by sequencing signals, were achieved. These latter recovery values reflect overall losses incurred in gel-focusing, gel-sizing, staining, destaining, high-pressure liquid extrusion, and N-terminal blockage. This work demonstrates that an array of protein spots can be systematically identified or defined by partial sequencing after high-pressure liquid extrusion from a 2-D gel matrix.(ABSTRACT TRUNCATED AT 250 WORDS)
DeWald
D B
DB
Biotechnology Research, Upjohn Company, Kalamazoo, Michigan 49001.
Pearson
J D
JD
D000180:Acrylic Resins; D000818:Animals; D001483:Base Sequence; D001923:Brain Chemistry; D002417:Cattle; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D007525:Isoelectric Focusing; D051379:Mice; D009419:Nerve Tissue Proteins / Q000302:isolation & purification*; D012967:Sodium Dodecyl Sulfate
D000180:Acrylic Resins; D009419:Nerve Tissue Proteins; C016680:polyacrylamide gels; D012967:Sodium Dodecyl Sulfate