Premium
Direct isolation of proteins from sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and analysis by electrospray‐ionization mass spectrometry
Author(s) -
Schuhmacher Martina,
Glocker Michael O.,
Wunderlin Markus,
Przybylski Michael
Publication year - 1996
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.1150170506
Subject(s) - electroelution , chemistry , chromatography , sodium dodecyl sulfate , electrospray ionization , gel electrophoresis , polyacrylamide gel electrophoresis , coomassie brilliant blue , mass spectrometry , molecular weight size marker , gel electrophoresis of proteins , molecular mass , two dimensional gel electrophoresis , dithiothreitol , elution , electrophoresis , biochemistry , proteomics , staining , medicine , pathology , gene , enzyme
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 β‐mercaptoethanol (β‐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.