Micropreparative gel electrophoresis of low-molecular-weight peptides: purification of highly insoluble amyloid peptide fragments.
We have used the continuous-elution micropreparative gel electrophoresis device described by Baumann and Lauraeus (Anal. Biochem. 214, 142-148, 1993) to purify low-molecular-weight peptide fragments from in-gel digested standard proteins as well as highly in-soluble amyloid peptides. Alzheimer's amyloid beta-peptide, gelsolin-derived amyloid peptide of the Finnish type, and a novel amyloid of the British type were purified from either homogenized brain or kidney tissue material to a high degree of purity in a single run. Using the high resolving capacity of the Tris-Tricine-SDS buffer system of Schaegger and von Jagow (Anal. Biochem. 166, 368-379, 1978) we were able to isolate two synthetic peptides with M(r)4329 and 3284, differing only by 1045 in mass. The total peptide recovery, as determined by amino acid sequence analysis and scanning densitometry, ranged between 60 and 80%. In order to demonstrate the utility of this technique we subjected some of the purified peptides to direct N-terminal amino acid sequence analysis, mass spectrometry, microbore high-performance liquid chromatography, and immunochemical studies. Our results show that micropreparative gel electrophoresis is an effective tool for the isolation of not only larger polypeptides but also small peptide fragments in a form suitable for further biological use.
Baumann
M
M
Department of Pathology, New York University Medical Center, New York 10016, USA. marc.baumann@helsinki.fi
Golabek
A
A
Lalowski
M
M
Wisniewski
T
T
D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D000682:Amyloid / Q000302:isolation & purification; D016229:Amyloid beta-Peptides / Q000302:isolation & purification*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D008832:Microchemistry / Q000379:methods; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000302:isolation & purification*; D010455:Peptides / Q000302:isolation & purification*; D012995:Solubility
D048868:Adaptor Proteins, Signal Transducing; D000682:Amyloid; D016229:Amyloid beta-Peptides; C120083:ITM2B protein, human; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D010446:Peptide Fragments; D010455:Peptides; C103598:amyloid AGel protein, human