A "fluorescence switch" technique increases the sensitivity of proteomic detection and identification of S-nitrosylated proteins.
Protein S-nitrosylation is a reversible post-translational modification of protein cysteines that is increasingly being considered as a signal transduction mechanism. The "biotin switch" technique marked the beginning of the study of the S-nitrosoproteome, based on the specific replacement of the labile S-nitrosylation by a more stable biotinylation that allowed further detection and purification. However, its application for proteomic studies is limited by its relatively low sensitivity. Thus, typical proteomic experiments require high quantities of protein extracts, which precludes the use of this method in a number of biological settings. We have developed a "fluorescence switch" technique that, when coupled to 2-DE proteomic methodologies, allows the detection and identification of S-nitrosylated proteins by using limited amounts of starting material, thus significantly improving the sensitivity. We have applied this methodology to detect proteins that become S-nitrosylated in endothelial cells when exposed to S-nitroso-L-cysteine, a physiological S-nitrosothiol, identifying already known S-nitrosylation targets, as well as proteins that are novel targets. This "fluorescence switch" approach also allowed us to identify several proteins that are denitrosylated by thioredoxin in cytokine-activated RAW264.7 (murine macrophage) cells. We believe that this method represents an improvement in order to approach the identification of S-nitrosylated proteins in physiological conditions.
Servicio de Inmunología, Hospital de La Princesa, Madrid, Spain.
Daniel
D
Tello
Carlos
C
Tarín
Patricia
P
Ahicart
Rosa
R
Bretón-Romero
Santiago
S
Lamas
Antonio
A
Martínez-Ruiz
D000818:Animals; D002460:Cell Line; D002478:Cells, Cultured; D003545:Cysteine / Q000031:analogs & derivatives* / Q000032:analysis / Q000378:metabolism; D042783:Endothelial Cells / Q000166:cytology; D005453:Fluorescence; D006801:Humans; D008264:Macrophages / Q000166:cytology; D051379:Mice; D015538:Nitrosation; D011499:Protein Processing, Post-Translational; D011506:Proteins / Q000032:analysis* / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*; D026403:S-Nitrosothiols / Q000032:analysis / Q000378:metabolism*; D012680:Sensitivity and Specificity
D011506:Proteins; D026403:S-Nitrosothiols; C021315:S-nitrosocysteine; D003545:Cysteine