High-resolution 2-DE for resolving proteins, protein adducts and complexes in plasma |
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Authors: | Candiano Giovanni Santucci Laura Petretto Andrea Pavone Barbara Del Boccio Piero Musante Luca Bruschi Maurizio Federici Giorgio Gusmano Rosanna Urbani Andrea Ghiggeri Gian M |
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Affiliation: | Laboratory on Pathophysiology of Uremia, G. Gaslini Children Hospital, Largo G. Gaslini, Genova, Italy. |
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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 beta), apolipoproteins (apolipoprotein B) and cell debris (vinculin, gelsolin, tropomyosin, dystrobrevin beta, fibrinectin I). Nat/SDS PAGE also allowed separation of nicked forms of albumin, Apo B100 and alpha2-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. |
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Keywords: | 2‐DE Albumin Nondenaturing electrophoresis Plasma proteins |
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