RESUMO
An automatic method for disulfide bond assignment using dimethyl labeling and computational screening of a(1) ions with customized software, RADAR, is developed. By utilization of the enhanced a(1) ions generated from labeled peptides, the N-terminal amino acids from disulfide-linked peptides can be determined. In this study, we applied this method for structural characterization of recombinant monoclonal antibodies, an important group of therapeutic proteins. In addition to a(1) ion screening and molecular weight match, new RADAR is capable of confirming the matched peptide pairs by further comparing the collision-induced dissociation (CID) fragment ions. With the N-terminal amino acid identities as a threshold, the identification of disulfide-linked peptide pairs can be achieved rapidly at a higher confidence level. Unlike most current approaches, prior knowledge of disulfide linkages or a high-end mass spectrometer is not required, and tedious work or deliberate interpretation can be avoided in this study. Our approach makes it possible to analyze unknown disulfide bonds of protein pharmaceuticals as well as their degraded forms without further protein separation. It can be used as a convenient quality examination tool during biopharmaceutical development and manufacturing processes.
Assuntos
Anticorpos Monoclonais/química , Dissulfetos/química , Proteínas Recombinantes/química , Software , Sequência de Aminoácidos , Química Farmacêutica , Humanos , Íons , Dados de Sequência Molecular , Espectrometria de Massas por Ionização por Electrospray , Coloração e Rotulagem , Espectrometria de Massas em TandemRESUMO
Intracerebral hemorrhage (ICH) is a common and severe neurological disorder associated with high morbidity and mortality rates. Despite extensive research into its pathology, there are no clinically approved neuroprotective treatments for ICH. Increasing evidence has revealed that inflammatory responses mediate the pathophysiological processes of brain injury following ICH. Experimental ICH was induced by direct infusion of 100 µL fresh (non-heparinized) autologous whole blood into the right basal ganglia of Sprague-Dawley rats at a constant rate (10 µL/min). The simvastatin group was administered simvastatin (15 mg/kg) and the combination therapy group was administered simvastatin (10 mg/kg) and ezetimibe (10 mg/kg). Magnetic resonance imaging (MRI), the forelimb use asymmetry test, the Morris water maze test, and two biomarkers were used to evaluate the effect of simvastatin and combination therapy. MRI imaging revealed that combination therapy resulted in significantly reduced perihematomal edema. Biomarker analyses revealed that both treatments led to significantly reduced endothelial inflammatory responses. The forelimb use asymmetry test revealed that both treatment groups had significantly improved neurological outcomes. The Morris water maze test revealed improved neurological function after combined therapy, which also led to less neuronal loss in the hippocampal CA1 region. In conclusion, simvastatin-ezetimibe combination therapy can improve neurological function, attenuate the endothelial inflammatory response and lead to less neuronal loss in the hippocampal CA1 region in a rat model of ICH.