Comprehensive identification of nitroxyl-reactive cysteines in human platelet proteins by quantitative mass spectrometry

• Main Author: Lin, Liwen
• Language: English
• Published: University of British Columbia 2011
• Online Access: http://hdl.handle.net/2429/37089

HNO, a small compound which can be produced upon hydrolysis of Angeli’s Salt, is known to be able to inhibit platelet aggregation. An important step to elucidate the mechanism of the antiaggregatory effect of HNO and define the drug’s targets is the identification of HNO-reactive proteins in platelets. The sulfhydryl group is considered the major target of HNO. It can be modified into either sulfinamide or disulfide. The sulfinamide modification is directly detectable by mass spectrometry; however, cysteines converted into disulfide groups by HNO cannot be monitored this way, because the origin of the other cysteine it reacts with is unknown. The goal of this thesis was to identify HNO-reactive cysteines in human platelet proteins regardless of the eventual modification states and discover cysteines which might play key roles in platelet inhibition by HNO. In the first series of studies, a differential alkylation method was developed, evaluated on a protein model and applied on human platelets to identify HNO-reactive cysteines. The results show that 32 HNO-reactive cysteines from 18 proteins were identified. Moreover, utilizing the detectable mass shift derived from sulfinamide, the modification states (i.e., sulfinamide or disulfide) of the 32 HNO-reactive cysteines were further investigated. In the second series of studies, isotope-coded affinity tag enrichment was combined with the differential alkylation method to increase the number of HNO-reactive cysteines discovered. The results show that 159 HNO-reactive cysteines from 78 proteins were identified. Studies have shown that platelet inhibition by HNO was time-dependent; the effect was not evident after 60 minutes of incubation with HNO. Therefore, a time-dependent study was performed to identify cysteines whose HNO-induced modifications were reversed after 60 minutes of incubation. The results show that the modifications of 83 cysteines out of the 159 HNO-reactive cysteines were reversible. Based on the reactivity toward HNO, the reversibility of HNO-induced modifications and the biological functions, talin, filamin, α-actinin and integrin αIIbβ3 are proposed as potential drug targets that may play key roles in platelet inhibition by HNO. === Science, Faculty of === Chemistry, Department of === Graduate