RT Journal Article
T1 A “fluorescence switch” technique increases the sensitivity of proteomic detection and identification of S‐nitrosylated proteins
A1 Tello, Daniel
A1 Tarín, Carlos
A1 Ahicart, Patricia
A1 Bretón‐Romero, Rosa
A1 Lamas, Santiago
A1 Martínez Ruiz, Antonio
AB 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 specificreplacement 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-activatedRAW264.7 (murine macrophage) cells. We believe that this method represents an improvement in order to approach the identification of S-nitrosylated proteins in physiological conditions.
SN 1615-9853
SN 1615-9861
YR 2009
FD 2009-12
LK https://hdl.handle.net/20.500.14352/104317
UL https://hdl.handle.net/20.500.14352/104317
LA eng
NO Gobierno de España
DS Docta Complutense
RD 28 jul 2024