title: New iodo-acetamido cyanines for labeling cysteine thiol residues. A strategy for evaluating plasma proteins and their oxido-redox status.
abstract: 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.
authors.affiliation: Division of Nephrology and Laboratory on Pathophysiology of Uremia, G Gaslini Children Hospital, Genoa, Italy.
authors.forename: Maurizio
authors.initials: M
authors.lastname: Bruschi
authors.forename: Stefano
authors.initials: S
authors.lastname: Grilli
authors.forename: Giovanni
authors.initials: G
authors.lastname: Candiano
authors.forename: Serena
authors.initials: S
authors.lastname: Fabbroni
authors.forename: Leopoldo
authors.initials: L
authors.lastname: Della Ciana
authors.forename: Andrea
authors.initials: A
authors.lastname: Petretto
authors.forename: Laura
authors.initials: L
authors.lastname: Santucci
authors.forename: Andrea
authors.initials: A
authors.lastname: Urbani
authors.forename: Rosanna
authors.initials: R
authors.lastname: Gusmano
authors.forename: Francesco
authors.initials: F
authors.lastname: Scolari
authors.forename: Gian Marco
authors.initials: GM
authors.lastname: Ghiggeri
mesh_terms: 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
chemical_list: D001798:Blood Proteins; D002232:Carbocyanines; D005456:Fluorescent Dyes; D013438:Sulfhydryl Compounds; D003545:Cysteine; D007460:Iodoacetamide