New iodo-acetamido cyanines for labeling cysteine thiol residues. A strategy for evaluating plasma proteins and their oxido-redox status

Maurizio Bruschi, Stefano Grilli, Giovanni Candiano, Serena Fabbroni, Leopoldo Della Ciana, Andrea Petretto, Laura Santucci, Andrea Urbani, Rosanna Gusmano, Francesco Scolari, Gian Marco Ghiggeri

Research output: Contribution to journalArticlepeer-review

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 H2O2 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 H2O2, C3NIASO3 and C5NIASO3 failed to detect any residual SH, implicating massive oxidation. In conclusion, this study describes the synthesis oftwo 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 oxidoredox status of proteins and were successfully utilized to extend the analysis of oxidation damage in patients with glomerulosclerosis.

Original languageEnglish
Pages (from-to)460-469
Number of pages10
JournalProteomics
Volume9
Issue number2
DOIs
Publication statusPublished - Jan 2009

Keywords

  • Cyanines
  • DIGE
  • Glomerulosclerosis
  • Oxido-redox status
  • Thiol reagents

ASJC Scopus subject areas

  • Molecular Biology
  • Biochemistry

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