Biosensor-Linked Immunosorbent Assay for the Quantification of Methionine Oxidation in Target Proteins

Abstract

Methionine oxidation is involved in regulating the protein activity and often leads to protein malfunction. However, tools for quantitative analyses of protein-specific methionine oxidation are currently unavailable. In this work, we developed a biological sensor that quantifies oxidized methionine in the form of methionine-R-sulfoxide in target proteins. The biosensor "tpMe-tROG" consists of methionine sulfoxide reductase B (MsrB), circularly permuted yellow fluorescent protein (cpYFP), thioredoxin, and protein G. Protein G binds to the constant region of antibodies against target proteins, specifically capturing them. Then, MsrB reduces the oxidized methionine in these proteins, leading to cpYFP fluorescence changes. We assessed this biosensor for quantitative analysis of methionine-R-sulfoxide in various proteins, such as calmodulin, IDLO, LegP, Sacde, and actin. We further developed an immunosorbent assay using the biosensor to quantify methionine oxidation in specific proteins such as calmodulin in animal tissues. The biosensor-linked immunosorbent assay proves to be an indispensable tool for detecting methionine oxidation in a protein-specific manner. This is a versatile tool for studying the redox biology of methionine oxidation in proteins.

Regulation of Photophysical Behaviors in Hyperbranched Aggregation-Induced Emission Polymers for Reactive Oxygen Species Scavenging

“…As an alternative to organic antioxidants, polymers bearing sulfur atoms have attracted particular attention. , The stable polymeric structures could prevent adverse effects and improve therapeutic efficacy, and the reductive thioether groups with hydrophobic-to-hydrophilic conversion when exposed to ROS make them ideal candidates for responsive drug delivery. In addition to various artificial thioethers, l -methionine is a kind of natural amino acid with excellent biocompatibility, and it can be oxidized to methionine sulfoxide by ROS and further reduced to methionine by methionine sulfoxide reductases in vivo . , Hence, developing an effective polymer nanomaterial with l -methionine as the scavenging active site is an effective approach for ROS treatment. Hyperbranched polymers have three-dimensional molecular configurations and abundant terminal groups, which provide sufficient grafting sites for l -methionine.…”

“…In addition to various artificial thioethers, L-methionine is a kind of natural amino acid with excellent biocompatibility, and it can be oxidized to methionine sulfoxide by ROS and further reduced to methionine by methionine sulfoxide reductases in vivo. 38,39 Hence, developing an effective polymer nanomaterial with Lmethionine as the scavenging active site is an effective approach for ROS treatment. Hyperbranched polymers have three-dimensional molecular configurations and abundant terminal groups, which provide sufficient grafting sites for Lmethionine.…”