Technology and methodology to manipulate proteins and study protein post-translation modifications

Abstract

Plants have intrinsic ROS/RNS metabolism and subsequent signaling to maintain the redox homeostasis. Redox post-translation modifications (PTMs) are mainly focus on the revisable cysteine modification due to the dual role of its protection proteins against irreversible modification and modulation of protein function. The global Redox PTMs study could provide insight into molecular mechanisms upon the external elicitors. In the present study, an in vivo quantitative thiol redox proteomic method, OxNSIL, has been developed, which combines chemical labeling by biotin-tagged alkylating reagents with heavy nitrogen stable isotope metabolic labeling in Arabidopsis to measure both the reduced and the oxidized cysteine thiol moieties in a single experiment. In order to validate the effectiveness of OxNSIL in the measurement of ROS-regulated thiol redox alteration, two external elicitors were applied onto Arabidopsis to trigger thiol redox state alteration. These results substantiate that the OxNSIL approach is useful in study of in planta redox alteration as well as ROS-induced cellular thiol redox state alterations within plant cells. Affinity chromatography is one of the fundamental and widely used technologies in the field of biological research field to manipulate tag containing biomolecules. The 3-D hierarchical sea-urchin-shaped α-Fe₂O₃ NWs, which offer a controlled microstructure and excellent stability for convenient storage, were functionalized with Ni-NTA via a general and efficient method, and showed higher histagged protein binding capacity than that of the same amount of commercial beads. The Ni-NTA functionalized silicon NW-chips conjugated with hexokinase I can be used to establish an artificial glycolytic pathway to perform the blood glucose degradation on-site. Phosphorylation, one of the important PTMs, was found to transduce ethylene signals in plants. To demonstrate the ethylene responsive phosphorylation of phosphosites identified from global quantitative phosphoproteomics of rcn1-1 mutant after 0 or 1-min of exogenous ethylene treatment, the selected peptides were quantified based on both dot blot and iTRAQ methods via the in vitro kinase assay. The consistent results of two quantification methods confirmed the peptides phosphorylation alteration according to ethylene treatment. Since the interaction of substrate and kinase is transient, it is still a change to find the interactive kinase. We developed a new strategy to capture the kinase of the peptide substrate through a photo-reactive crosslinker, Benzophenone-4-maleimide (BPM). In the assay condition, the affinity between BPM modified peptide and kinase makes them being close to each other and can be covalently linked together when activated with the bezophenone.

Date of Award	2014
Original language	English
Awarding Institution	The Hong Kong University of Science and Technology