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Saturday, January 3, 2009

Identification of nitroxyl-induced modifications in human platelet proteins using a novel mass spectrometric detection method
MCP Papers in Press. Published on December 31, 2008

Nitroxyl (HNO) exhibits many important pharmacological effects, including inhibition of platelet aggregation, and the HNO donor, Angeli’s salt (AS), has been proposed as a potential therapeutic agent in the treatment of disease including heart failure and alcoholism. Despite this, little is known about HNO’s mechanism of action, and its effects are rarely linked to specific protein targets of HNO or to the actual chemical changes that proteins undergo when in contact with HNO.

Here, we study the presumed major molecular target of HNO within the body: protein thiols.

Cysteine-containing tryptic peptides were reacted with HNO, generating the sulfinamide modification, and to a lesser extent, disulfide linkages. The sulfinamide modification was subjected to a comprehensive analysis including MS/MS by collision induced dissociation (CID) and electron capture dissociation (ECD), and MS3.

These studies revealed a characteristic neutral loss of HS(O)NH2 (65 Da) that is liberated from the modified cysteine upon CID, and can be monitored by mass spectrometry. Upon storage, partial conversion of the sulfinamide to sulfinic acid was observed, leading to coinciding neutral losses of 65 Da and 66 Da (HS(O)OH). Validation of the method was conducted using a targeted study of nitroxylated glyceraldehyde 3-phosphate dehydrogenase (GAPDH) extracted from AS-treated human platelets.

In these ex vivo experiments, the sample preparation process resulted in complete conversion of sulfinamide to sulfinic acid, making this the sole subject of further ex vivo studies.

A global proteomic analysis to discover platelet proteins that carry nitroxyl-induced modifications and a mass spectrometric HNO dose response analysis of the modified proteins was conducted to gain insight into the specificity and selectivity of this modification.

This identified 10 proteins that are modified dose dependently in response to HNO, providing for the first time a possible mechanistic link between HNO-induced modification and the physiological effects of HNO donors in platelets.

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