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

被引：20

作者：

Bruschi, Maurizio ^{[1
,2
,3
]}

Grilli, Stefano ^{[4
]}

Candiano, Giovanni ^{[1
,2
]}

Fabbroni, Serena ^{[4
]}

Della Ciana, Leopoldo ^{[4
]}

Petretto, Andrea ^{[5
]}

Santucci, Laura ^{[1
,2
,3
]}

Urbani, Andrea ^{[6
]}

Gusmano, Rosanna ^{[3
]}

Scolari, Francesco ^{[8
]}

Ghiggeri, Gian Marco ^{[1
,2
,7
]}

机构：

[1] G Gaslini Children Hosp, Lab Pathophysiol Uremia, I-16148 Genoa, Italy

[2] G Gaslini Children Hosp, Div Nephrol, I-16148 Genoa, Italy

[3] RenalChild Fdn, Genoa, Italy

[4] Cyanagen, Bologna, Italy

[5] G Gaslini Children Hosp, Mass Spectrometry Core Facil, I-16148 Genoa, Italy

[6] Univ G dAnnunzio, Dept Biomed Sci, Chieti, Italy

[7] G Gaslini Children Hosp, Dept Nephrol, I-16148 Genoa, Italy

[8] Univ Brescia, Chair Nephrol, Brescia, Italy

来源：

PROTEOMICS | 2009年 / 9卷 / 02期

关键词：

Cyanines; DIGE; Glomerulosclerosis; Oxido-redox status; Thiol reagents; DIFFERENCE GEL-ELECTROPHORESIS; HUMAN SERUM-ALBUMIN; PROTEOMIC ANALYSIS; FLUORESCENT DYES; MYELOPEROXIDASE; EXPRESSION; OXIDATION; ACID;

D O I：

10.1002/pmic.200800254

中图分类号：

Q5 [生物化学];

学科分类号：

071010 ; 081704 ;

摘要：

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 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.

引用

页码：460 / 469

页数：10

共 20 条

[1] A novel experimental design for comparative two-dimensional gel analysis: Two-dimensional difference gel electrophoresis incorporating a pooled internal standard [J].