High-resolution 2-DE for resolving proteins, proteins adducts and complexes in plasma

Giovanni Candiano, Laura Santucci, Andrea Petretto, Barbara Pavone, Piero Del Boccio, Luca Musante, Maurizio Bruschi, Giorgio Federici, Rosanna Gusmano, Andrea Urbani, Gian M. Ghiggeri

Research output: Contribution to journalArticle

Abstract

A 2-DE system has been devised in which proteins are first separated in their native state followed by separation according to mass under denaturing conditions (Nat/SDS-PAGE). Hydrophilic properties of the gel and the presence of dihydroxybisacrylamide in the first dimension allowed a good resolution for high-molecular-weight proteins and maintained interactions. With this method 252 plasma spots have been resolved and 140 have been characterized by MS as isoforms of 60 proteins, a relevant part of which (12) were not detected by traditional 2-D gels or by other nondenaturing 2-D techniques. The list includes complement factors (C4d, C7), coagulation factors (coagulation factor II, fibrin β), apolipoproteins (apolipoprotein B) and cell debris (vinculin, gelsolin, tropomyosin, dystrobrevin β, fibrinectin I). Nat/SDS PAGE also allowed separation of nicked forms of albumin, Apo B100 and α2-macroglobulin and showed the presence of atypical albumin adducts corresponding to post-translational and oxidation products. Our system provides therefore new tools for resolving proteins, protein aggregates and complexes and amplifies the potentiality of traditional electrophoretic analysis.

Original languageEnglish
Pages (from-to)682-694
Number of pages13
JournalElectrophoresis
Volume29
Issue number3
DOIs
Publication statusPublished - Feb 2008

Keywords

  • 2-DE
  • Albumin
  • Nondenaturing electrophoresis
  • Plasma proteins

ASJC Scopus subject areas

  • Clinical Biochemistry

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