Profiling of sickle red blood cell membrane proteins after in vitro exposure to N-acetylcysteine using isotope-coded affinity tag reagents and liquid chromatography tandem mass spectrometry

Sickle Cell Disease (SCD), characterized by sickle-shaped red blood cells (RBCs), is a genetic disorder caused by an amino acid substitution in the beta chain of hemoglobin. This abnormality can result in painful episodes, serious infections, chronic anemia, and organ damage in the patient [48]. N-acetylcysteine (NAC), an antioxidant that is a precursor in the biosynthesis of reduced glutathione (GSH), has been previously shown to decrease the formation of high density irreversible sickle cells (ISCs) in vitro [16]. In another in vivo study, NAC was observed to lower the number of vaso-occlusive episodes in participants [35]. The purpose of this study was to construct a quantitative profile of untreated versus NAC-treated sickle RBC membrane proteins to decipher whether proteins change significantly due to in vitro exposure to the drug. Two studies were implemented: a technique variation study to determine the variations in the procedure and a drug effect study to analyze the effects of 1mM, 10mM, and 20mM NAC on sickle membrane proteins. Profiling of the RBC membrane proteins was accomplished using cleavable isotope-coded affinity tag (cICAT) reagents and liquid chromatography tandem mass spectrometry (muLC/MS/MS). Using the ANOVA model, the variation inherent in the method, as measured by the standard deviation, was estimated to be approximately 20% on the ratio scale. Changes in ratios of more than two standard deviations, which in this case is around 40%, can be considered statistically significant. None of the sickle membrane proteins showed a statistically significant change in mean ratios after the addition of the three different NAC concentrations. These results lead to the conclusion that there are no changes in the sickle RBC membrane proteins due to the presence of NAC.