Direct isolation of proteins from sodium dodecyl sulfate-polyacrylamide gel electrophoresis and analysis by electrospray-ionization mass spectrometry.
A new method for the isolation of proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), using electroelution with a modified buffer system, is described. The method is suitable for direct characterization by electrospray-ionization mass spectrometry (ESI-MS), or alternatively matrix-assisted laser desorption-ionization (MALDI), no additional purification steps being required. Following separation by SDS-PAGE, proteins were stained with Coomassie Blue, and isolated by electroelution using SDS-free ammonium acetate (pH 2.5) as elution buffer. The polarity of the electroelution system was reversed, which, upon in situ dissociation of protein-SDS complexes, resulted in migration of free proteins to the cathode under the conditions employed. Recovery rates of 25-58% were determined for model proteins. Analyses by ESI-MS provided exact molecular weight determinations of isolated proteins and of protein mixtures not resolved by SDS-PAGE. Essentially SDS-free molecular ions were obtained, except for bovine serum albumin with one SDS-adduct. Charge distribution of molecular ions were similar to those of the native proteins. The effects of beta-mercaptoethanol (beta-ME) and dithiothreitol (DTT) during electrophoresis were studied with hen egg white lysozyme, revealing the formation of mixed disulfide adducts between proteins and reducing agents. In a first bioanalytical application, hemofiltrate proteins from a patient with uremia were separated by SDS-PAGE. An ESI-MS analysis of the proteins isolated from the two most intensive gel bands enabled exact molecular weight determinations, and demonstrated that a gel band of ca. 17 kDa consisted of two different proteins.
Schuhmacher
M
M
Faculty of Chemistry, University of Konstanz, Germany.
Glocker
M O
MO
Wunderlin
M
M
Przybylski
M
M
D000595:Amino Acid Sequence; D000818:Animals; D001798:Blood Proteins / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D005782:Gels; D006801:Humans; D007328:Insulin / Q000032:analysis; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D009113:Muramidase / Q000032:analysis; D009211:Myoglobin / Q000032:analysis; D011506:Proteins / Q000302:isolation & purification*; D012710:Serum Albumin, Bovine / Q000032:analysis; D012967:Sodium Dodecyl Sulfate
D001798:Blood Proteins; D005782:Gels; D007328:Insulin; D009211:Myoglobin; D011506:Proteins; D012710:Serum Albumin, Bovine; D012967:Sodium Dodecyl Sulfate; D009113:Muramidase