RESUMO
Micronutrient deficiencies are common in locales where people must rely upon sorghum as their staple diet. Sorghum grain is seriously deficient in provitamin A (ß-carotene) and in the bioavailability of iron and zinc. Biofortification is a process to improve crops for one or more micronutrient deficiencies. We have developed sorghum with increased ß-carotene accumulation that will alleviate vitamin A deficiency among people who rely on sorghum as their dietary staple. However, subsequent ß-carotene instability during storage negatively affects the full utilization of this essential micronutrient. We determined that oxidation is the main factor causing ß-carotene degradation under ambient conditions. We further demonstrated that coexpression of homogentisate geranylgeranyl transferase (HGGT), stacked with carotenoid biosynthesis genes, can mitigate ß-carotene oxidative degradation, resulting in increased ß-carotene accumulation and stability. A kinetic study of ß-carotene degradation showed that the half-life of ß-carotene is extended from less than 4 wk to 10 wk on average with HGGT coexpression.
Assuntos
Alimentos Fortificados , Sorghum/metabolismo , Vitamina E/metabolismo , beta Caroteno/metabolismo , Cromatografia Líquida de Alta Pressão , DNA Bacteriano/genética , Endosperma/metabolismo , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Sementes/metabolismo , Sorghum/enzimologia , Sorghum/genéticaRESUMO
BACKGROUND: Liquid chromatography coupled with tandem mass spectrometry is increasingly used for protein detection for transgenic crops research. Currently this is achieved with protein reference standards which may take a significant time or efforts to obtain and there is a need for rapid protein detection without protein reference standards. RESULTS: A sensitive and specific method was developed to detect target proteins in transgenic maize leaf crude extract at concentrations as low as â¼30 ng mg(-1) dry leaf without the need of reference standards or any sample enrichment. A hybrid Q-TRAP mass spectrometer was used to monitor all potential tryptic peptides of the target proteins in both transgenic and non-transgenic samples. The multiple reaction monitoring-initiated detection and sequencing (MIDAS) approach was used for initial peptide/protein identification via Mascot database search. Further confirmation was achieved by direct comparison between transgenic and non-transgenic samples. Definitive confirmation was provided by running the same experiments of synthetic peptides or protein standards, if available. CONCLUSION: A targeted proteomic mass spectrometry method using MIDAS approach is an ideal methodology for detection of new proteins in early stages of transgenic crop research and development when neither protein reference standards nor antibodies are available. © 2016 Society of Chemical Industry.
Assuntos
Folhas de Planta/química , Proteínas de Plantas/química , Zea mays/química , Cromatografia Líquida de Alta Pressão , Humanos , Engenharia de Proteínas , Proteômica , Valores de Referência , Sensibilidade e Especificidade , Espectrometria de Massas em TandemRESUMO
Development of monoclonal and bispecific antibody-based protein therapeutics requires detailed characterization of native disulfide linkages, which is commonly achieved through peptide mapping under non-reducing conditions followed by liquid chromatography-mass spectrometry (LC-MS) analysis. One major challenge of this method is incomplete protein digestion due to insufficient denaturation of antibodies under non-reducing conditions. For a long time, researchers have explored various strategies with the aim of efficiently digesting antibody drugs when the disulfide bonds remain intact, but few could achieve this by using a simple and generic approach with well controlled disulfide scrambling artifacts. Here, we report a simple method for fast and efficient mapping of native disulfides of monoclonal and bispecific antibody-based protein therapeutics. The method was optimized to achieve optimal digestion efficiency by denaturing proteins with 8â¯M urea plus 0-1.25â¯M guanidine-HCl at elevated temperature (50 °C), followed by two-step digestion with trypsin/Lys-C mix using a one-pot reaction. The only parameter that needs to be optimized for different proteins is the concentration of guanidine-HCl present. This simplified sample preparation eliminated buffer exchange and can be completed within three hours. By using this new method, all native disulfide bonds were confirmed for these monoclonal and bispecific antibodies with high confidence. When compared with a commercial kit utilizing low-pH digestion condition, the new method demonstrated higher digestion efficiency and shorter sample preparation time. These results suggest this new one-pot-two-step digestion method is suitable for the characterization of antibody disulfide bonds, particularly for those antibodies with digestion-resistant domains under typical digestion conditions.