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Redox Proteomic Analysis Reveals Microwave-Induced Oxidation Modifications of Myofibrillar Proteins from Silver Carp (Hypophthalmichthys molitrix)

Title: Redox Proteomic Analysis Reveals Microwave-Induced Oxidation Modifications of Myofibrillar Proteins from Silver Carp (Hypophthalmichthys molitrix)

Journal: Journal of Agricultural and Food Chemistry

Published: 2021

Background

In the field of food science, protein oxidation modifications directly affect its structure, function, and nutritional value, especially the oxidation state of myofibrillar proteins (such as myosin and actin), which are closely related to food texture and gel properties. Microwave heating, as an efficient and rapid food processing technology, has been widely used in the food industry. However, microwave heating may induce protein oxidation modifications, thereby affecting its functional characteristics. Currently, the specific mechanisms of microwave heating on protein oxidation modifications and their impact on protein functions are not yet clear. This study aims to systematically analyze the effects of microwave heating on the oxidation modifications of myofibrillar proteins in silver carp using redox proteomics technology, revealing its molecular mechanisms and functional significance.

Materials & Methods

Extraction of MPs

Muscles immersed with 10 times the volume (v/w) of low-salt buffer A (0.05 M KCl, 20 mM Tris–HCl, 2 mM MgCl2, and 1 mM EGTA, pH 7.0) were homogenized, followed by centrifugation at 5000×g for 20 min at 4℃. This process was repeated three times to remove the water-soluble proteins. The precipitate, which contained the target MPs, was extracted at 4℃ with 4 times the volume (v/w) of high-salt buffer (0.60 M KCl, 20 mM Tris–HCl, pH 6.8) for 6 h. After filtering the connective tissue with a gauze, the sample was centrifuged at 12,000g for 20 min, and the supernatant was treated with deionized water at 4℃ to precipitate the MPs. Finally, the MP pellets were collected and kept on ice for 24 h.

Protein Preparation and iodoTMT Labeling

The general strategy was as follows: the unmodified sulfhydryl groups on proteins were first blocked by IAM. Subsequently, all the oxidized cysteines were reduced by tris(2-carboxyethyl)phosphine (TCEP), and the reduced cysteines were labeled with iodoTMT. Finally, the labeled peptides were enriched for proteomic analysis.

Redox proteomic service Figure 1: Experimental principle and workflow of redox proteomic analysis of MPs using iodoTMT labeling.

Results

The study found that microwave heating significantly increased the oxidation modifications of cysteine residues in myofibrillar proteins (such as disulfide bonds, S-glutathionylation, etc.). Key oxidation sites include Cys9 of myosin heavy chain and Cys783 of actin. Oxidation modifications at these sites may affect the protein's conformation and function. Microwave heating significantly enhanced the gel strength and water-holding capacity of myofibrillar proteins by promoting oxidative crosslinking at the Cys9 site of myosin. Compared to traditional water bath heating, microwave heating induced more significant protein oxidation modifications in a shorter time.

Redox proteomic service Figure 2. (A) SDS-PAGE patterns of MPs under reducing and nonreducing conditions. M: marker, lines 1–3: UH, WB, and MW treatments under nonreducing conditions; lines 4–6: UH, WB, and MW treatments under reducing conditions; MHC: myosin heavy chain, CLP: cross-linking proteins. (B) Number of oxidative sites per protein in samples. (C) Hierarchical cluster analysis of the differential proteins of UH, WB, and MW. (D) Most conserved motifs of redox-modified cysteine. (E) Motif analysis of the differentially quantified proteins.

Redox proteomic service Figure 3. GO analysis of the oxidized proteins. The X-axis indicates the GO terms and the Y-axis indicates the number of proteins.

Redox proteomic service Figure 4. Main functional classification with the most significant enrichment of the differential proteins. (A) BP, (B) CC, and (C) MF.

Redox proteomic service Figure 5. Hierarchical cluster analysis of the enrichment level of GO functions terms. (A) BP, (B) CC, and (C) MF.

Redox proteomic service Figure 6. PPI analysis of the differentially expressed proteins in (A) WB/UH and (B) MW/UH.

Reference

Jiao, Xidong et al. "Redox Proteomic Analysis Reveals Microwave-Induced Oxidation Modifications of Myofibrillar Proteins from Silver Carp (Hypophthalmichthys molitrix)." Journal of agricultural and food chemistry vol. 69,33 (2021): 9706-9715. https://doi.org/10.1021/acs.jafc.1c03045

* For Research Use Only. Not for use in diagnostic procedures.

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