Abstract 18132: Identification of the Myocardial S-nitrosothiol Proteome
Myocardial ischemic preconditioning (IPC) is associated with an increase in protein S-nitrosothiol (SNO) formation. Furthermore, perfusion with the S-nitrosylating agent GSNO has been shown to increase protein SNO and is cardioprotective. SNO is a reversible protein modification that is thought to provide cardioprotection during ischemia-reperfusion (IR) injury by targeting critical proteins. In order to characterize the cardiac SNO proteome, we utilized SNO-resin assisted capture (SNO-RAC) with mass spectrometry to identify SNO proteins and determine the site of SNO. A modified biotin switch was performed using control whole heart homogenates (60′ perfusion) incubated with or without GSNO. SNO-RAC analysis identified 433 unique SNO proteins in GSNO treated homogenates, many of which contained multiple SNO sites. These proteins represent the SNO proteome and show the potential for endogenous SNO formation. To determine changes in the SNO proteome associated with cardioprotection, we also examined homogenates from hearts subjected to IPC (4 cycles IPC), IR (60′ perfusion, 20′ ischemia, 5′ reperfusion), and IPC-IR (20′ perfusion, 4 cycles IPC, 20′ ischemia, 5′ reperfusion). Of the 433 SNO proteins identified with GSNO treatment, 27 were identified to be endogenously SNO in control homogenates in the absence of any treatment. Interestingly, SNO-RAC analysis identified 79 SNO proteins with IPC. These SNO proteins included the F1F0 ATPase, voltage-dependent anion-selective channel protein 2, peptidyl-prolyl cis-trans isomerase, and aldehyde dehydrogenase 2. Several endogenous SNO proteins also showed increased SNO with IPC, including GAPDH, malate dehydrogenase, aconitase, SERCA2a, and α-ketoglutarate dehydrogenase (SNO increased at same site for each respective protein). Following IR, the number of SNO proteins identified via SNO-RAC decreased to endogenous levels with IPC-IR (11 proteins), indicating that SNO formation is quickly reversible. SNO-RAC identified a level of SNO proteins similar to endogenous levels with IR alone (33 proteins). Therefore, this study provides novel information regarding the cardiac SNO proteome, as well as additional information on the sites of SNO for key proteins involved in cardioprotection.
- © 2010 by American Heart Association, Inc.