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

Human plasma proteins were separated by combining four types of two-dimensi onal electrophoresis (2-DE) techniques to obtain systematic information on proteins and their constituent polypeptides. A micro gel system was employe d to facilitate the analysis. A plasma sample was first analyzed under nond enaturing conditions of electrophoresis (Type I 2-DE) to characterize the p roperties of proteins under physiological conditions. The sample was then a nalyzed, employing nondenaturing isoelectric focusing in the first dimensio n and sodium dodecyl sulfate (SDS) electrophoresis in the second dimension (Type II 2-DE), to study the dissociation of noncovalently bound protein su bunits. 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 t he dissociation of disulfide-bonded polypeptides. In the fourth type of 2-D E (Type IV 2-DE), the conditions of denaturing 2-DE were employed; the samp le was treated with SDS-mercaptoethanol-urea-Nonidet P-40, separated by den aturing isoelectric focusing, and then subjected to SDS electrophoresis. Th e combined 2-DE technique will be useful to construct a comprehensive datab ase of plasma proteins combining a "nondenaturing protein map" (a protein m ap) and a "denaturing protein map" (a polypeptide map).