A nondenaturing protein map of human plasma proteins correlated with a denaturing polypeptide map combining techniques of micro two-dimensional gel electrophoresis.

Abstract

Human plasma proteins were separated by combining four types of two-dimensional electrophoresis (2-DE) techniques to obtain systematic information on proteins and their constituent polypeptides. A micro gel system was employed to facilitate the analysis. A plasma sample was first analyzed under nondenaturing conditions of electrophoresis (Type I 2-DE) to characterize the properties of proteins under physiological conditions. The sample was then analyzed, employing nondenaturing isoelectric focusing in the first dimension and sodium dodecyl sulfate (SDS) electrophoresis in the second dimension (Type II 2-DE), to study the dissociation of noncovalently bound protein subunits. In the third type of 2-DE (Type III 2-DE), proteins were separated by nondenaturing isoelectric focusing and treated with urea/mercaptoethanol/SDS and then subjected to second-dimension SDS electrophoresis, to study the dissociation of disulfide-bonded polypeptides. In the fourth type of 2-DE (Type IV 2-DE), the conditions of denaturing 2-DE were employed; the sample was treated with SDS-mercaptoethanol-urea-Nonidet P-40, separated by denaturing isoelectric focusing, and then subjected to SDS electrophoresis. The combined 2-DE technique will be useful to construct a comprehensive database of plasma proteins combining a "nondenaturing protein map" (a protein map) and a "denaturing protein map" (a polypeptide map).