High-resolution 2-DE for resolving proteins, protein adducts and complexes in plasma.
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.
Candiano
Giovanni
G
Laboratory on Pathophysiology of Uremia, G. Gaslini Children Hospital, Largo G. Gaslini, Genova, Italy.
Santucci
Laura
L
Petretto
Andrea
A
Pavone
Barbara
B
Del Boccio
Piero
P
Musante
Luca
L
Bruschi
Maurizio
M
Federici
Giorgio
G
Gusmano
Rosanna
R
Urbani
Andrea
A
Ghiggeri
Gian M
GM
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
D016632:Apolipoprotein A-I; D053299:Apolipoprotein B-100; D001798:Blood Proteins; D006242:Haptoglobins; D046912:Multiprotein Complexes; D011228:Prealbumin; D012709:Serum Albumin; D000511:alpha-Macroglobulins