New iodo-acetamido cyanines for labeling cysteine thiol residues. A strategy for evaluating plasma proteins and their oxido-redox status.
Two new iodoacetamide-substituted cyanines, C3NIASO3 and C5NIASO3, were synthesized starting from hemicyanine and were utilized for labeling plasma proteins. Specificity, sensitivity and feasibility for SH residues was tested utilizing an equimolar mixture of standard proteins and with normal plasma. Oxidized plasma proteins following H(2)O(2 )exposure and plasma from patients with focal glomerulosclerosis were analyzed as models of altered protein oxido-redox status. Following optimization of the assay (dye/protein ratio, pH), C3NIASO3 and C5NIASO3 gave a sensitivity slightly better than N-hydroxysuccinimidyl dyes for plasma proteins and were successfully employed for differential display electrophoresis (DIGE). Twenty-nine proteins were detected in normal plasma after 2-DE while less proteins were detected in plasma of patients with glomerulosclerosis. Following massive 'in vitro' oxidation with H(2)O(2), C3NIASO3 and C5NIASO3 failed to detect any residual SH, implicating massive oxidation. In conclusion, this study describes the synthesis of two new iodoacetamide cyanines that can be utilized for the analysis of plasma proteins with 2-DE and DIGE. They are also indicated for the definition of the oxido-redox status of proteins and were successfully utilized to extend the analysis of oxidation damage in patients with glomerulosclerosis.
Bruschi
Maurizio
M
Division of Nephrology and Laboratory on Pathophysiology of Uremia, G Gaslini Children Hospital, Genoa, Italy.
Grilli
Stefano
S
Candiano
Giovanni
G
Fabbroni
Serena
S
Della Ciana
Leopoldo
L
Petretto
Andrea
A
Santucci
Laura
L
Urbani
Andrea
A
Gusmano
Rosanna
R
Scolari
Francesco
F
Ghiggeri
Gian Marco
GM
D001798:Blood Proteins / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism; D002232:Carbocyanines / Q000737:chemistry*; D003545:Cysteine / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005456:Fluorescent Dyes / Q000737:chemistry*; D005923:Glomerulosclerosis, Focal Segmental / Q000097:blood / Q000378:metabolism; D006801:Humans; D007460:Iodoacetamide / Q000737:chemistry*; D010084:Oxidation-Reduction; D040901:Proteomics / Q000379:methods; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013438:Sulfhydryl Compounds / Q000378:metabolism
D001798:Blood Proteins; D002232:Carbocyanines; D005456:Fluorescent Dyes; D013438:Sulfhydryl Compounds; D003545:Cysteine; D007460:Iodoacetamide