title: High-resolution 2-DE for resolving proteins, protein adducts and complexes in plasma.
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.
authors.affiliation: Laboratory on Pathophysiology of Uremia, G. Gaslini Children Hospital, Largo G. Gaslini, Genova, Italy.
authors.forename: Giovanni
authors.initials: G
authors.lastname: Candiano
authors.forename: Laura
authors.initials: L
authors.lastname: Santucci
authors.forename: Andrea
authors.initials: A
authors.lastname: Petretto
authors.forename: Barbara
authors.initials: B
authors.lastname: Pavone
authors.forename: Piero
authors.initials: P
authors.lastname: Del Boccio
authors.forename: Luca
authors.initials: L
authors.lastname: Musante
authors.forename: Maurizio
authors.initials: M
authors.lastname: Bruschi
authors.forename: Giorgio
authors.initials: G
authors.lastname: Federici
authors.forename: Rosanna
authors.initials: R
authors.lastname: Gusmano
authors.forename: Andrea
authors.initials: A
authors.lastname: Urbani
authors.forename: Gian M
authors.initials: GM
authors.lastname: Ghiggeri
mesh_terms: D000328:Adult; D016632:Apolipoprotein A-I / Q000097:blood / Q000302:isolation & purification; D053299:Apolipoprotein B-100 / Q000097:blood / Q000302:isolation & purification; D001797:Blood Protein Electrophoresis / Q000379:methods*; D001798:Blood Proteins / Q000737:chemistry / Q000302:isolation & purification*; D015153:Blotting, Western; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D005260:Female; D006242:Haptoglobins / Q000302:isolation & purification; D006801:Humans; D008297:Male; D008875:Middle Aged; D046912:Multiprotein Complexes; D011228:Prealbumin / Q000302:isolation & purification; D012709:Serum Albumin / Q000302:isolation & purification; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013194:Staining and Labeling; D053719:Tandem Mass Spectrometry; D000511:alpha-Macroglobulins / Q000302:isolation & purification
chemical_list: D016632:Apolipoprotein A-I; D053299:Apolipoprotein B-100; D001798:Blood Proteins; D006242:Haptoglobins; D046912:Multiprotein Complexes; D011228:Prealbumin; D012709:Serum Albumin; D000511:alpha-Macroglobulins