Differential proteome analysis of serum proteins associated with the development of type 2 diabetes mellitus in the KK-Ay mouse model using the iTRAQ technique
ABSTRACT To identify candidate serum molecules associated with the progression of type 2 diabetes mellitus (T2DM), we carried out differential proteomic analysis using the KK-Ay mouse, an animal model of T2DM with obesity. We employed an iTRAQ-based quantitative proteomic approach to analyze the proteomic changes in the sera collected from a pair of 4-week-old KK-Ay versus C57BL/6 mice. Among the 227 proteins identified, a total of 45 proteins were differentially expressed in KK-Ay versus C57BL/6 mice. We comparatively analyzed a series of the sera collected at 4 and 12 weeks of age from KK-Ay and C57BL/6 mice for the target protein using multiple reaction monitoring analysis, and identified 8 differentially expressed proteins between the sera of these mice at both time points. Among them, serine (or cysteine) peptidase inhibitor, clade A, member 3K (SERPINA3K) levels were elevated significantly in the sera of KK-Ay mice compared to C57BL/6 mice. An in vitro assay revealed that the human homologue SERPINA3 increased the transendothelial permeability of retinal microvascular endothelial cells, which may be involved in the pathogenesis of diabetes and/or diabetic retinopathy. With the identified proteins, our proteomics study could provide valuable clues for a better understanding of the underlying mechanisms associated with T2DM.
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ABSTRACT: Benzothiadiazole (BTH) is an artificial inducer of systemic acquired resistance. Due to rice being an important food crop and model plant, we investigated its response to BTH using label-free proteomics technology coupled with bioinformatics. Protein expression levels were verified using the multi-reaction monitoring mode and semi-quantitative RT-PCR. BTH treatment can up-or down-regulate many proteins produced by the rice host at all four periods, with the numbers of proteins being 6/24, 9/10, 14/10, and 8/20, respectively. Compared with mock treat-ments (phosphate buffered saline with 0.1 % dimethylsulf-oxide and 0.5 % Tween-20), some proteins related to plant resistance were only detected after BTH treatments, such as ascorbate peroxidase (POD) 3, chitinase A, thioredoxin-dependent POD 2, beta-1,3-glucanase 2, POD superfamily protein, major facilitator superfamily (MFS) protein, cop-per/zinc-superoxide dismutase (SOD) 1, pathogenesis-related protein (PR) 1. Other proteins showing up-regulation after BHT treatment included PR-5, glyceraldehyde-3-phosphate dehydrogenase C, plasma-membrane associated cation-binding protein 1, and oxidoreductase family pro-teins. These results indicated that BTH was involved with inducing rice resistance. Some up-regulated proteins were also involved in other metabolic processes. The activity and expression level of POD, phenylalanine ammonia-lyase (PAL), and SOD, lipoxygenase (LOX), beta-1,3-glucanas-es, and chitinases were determined using the enzyme activity assay and semi-quantitative RT-PCR. These results indicated that BTH can enhance the activity of beta-1,3-glucanases, LOX, PAL, and POD. BTH can also induce up-regulation of the copper/zinc-SOD, ascorbate POD, gluta-thione POD 1, Chitinase, and LOX1 genes. Keywords Benzothiadiazole (BTH) Á Label-free proteomics Á Bioinformatics Á Multi-reaction monitoring (MRM) Á Protein expression Á Enzyme activity Á Semi-quantitative RT-PCRJournal of Plant Growth Regulation 01/2015; 34(2). DOI:10.1007/s00344-015-9476-y · 2.06 Impact Factor