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

ArticleinJournal of proteomics · March 2013with19 Reads
DOI: 10.1016/j.jprot.2013.03.014 · Source: PubMed
Abstract
Unlabelled: To identify candidate serum molecules associated with the progression of type 2 diabetes mellitus (T2DM), we carried out differential proteomic analysis using the KK-A(y) 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-A(y) versus C57BL/6 mice. Among the 227 proteins identified, a total of 45 proteins were differentially expressed in KK-A(y) versus C57BL/6 mice. We comparatively analyzed a series of the sera collected at 4 and 12weeks of age from KK-A(y) 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-A(y) 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. Biological significance: In this paper, we investigated the serum proteome of KK-A(y) mice in a pre-diabetic state compared to that of wild type controls in an attempt to uncover early diagnostic markers of diabetes that are maintained through a diabetic phenotype. We used iTRAQ-based two-dimensional LC-MS/MS serum profiling, and identified several differentially expressed proteins at the pre-diabetic stage. The differential expression was confirmed by multiple reaction monitoring assay, which is fast gaining ground as a sensitive, specific, and cost-effective methodology for relative quantification of the candidate proteins. Using these techniques, we have identified eight candidate proteins of interest including SERPINA3K, which may be important in the pathology of T2DM and/or diabetic retinopathy.
    • "Once the type of sample to be used has been selected, it is necessary to optimize the protocol for tissue disruption/cell-lysis or for the isolation of the proteins from body fluids using commercial reagents including protease and phosphatase inhibitors. When analysing serum/plasma, it may be necessary to use an albumin/IgG depletion kit to identify proteins whose concentration is low [15]. We have found that many proteins do not exist as a single form but as isoforms probably generated by post-translational modifications (PTM) of a given protein. "
    [Show abstract] [Hide abstract] ABSTRACT: The incidence of obesity and type diabetes 2 has increased dramatically resulting in an increased interest in its biomedical relevance. However, the mechanisms that trigger the development of diabetes type 2 in obese patients remain largely unknown. Scientific, clinical and pharmaceutical communities are dedicating vast resources to unravel this issue by applying different omics tools. During the last decade, the advances in proteomic approaches and the Human Proteome Organization have opened and are opening a new door that may be helpful in the identification of patients at risk and to improve current therapies. Here, we briefly review some of the advances in our understanding of type 2 diabetes that have occurred through the application of proteomics. We also review, in detail, the current improvements in proteomic methodologies and new strategies that could be employed to further advance our understanding of this pathology. By applying these new proteomic advances, novel therapeutic and/or diagnostic protein targets will be discovered in the obesity/Type 2 diabetes area. © 2015 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.
    Article · May 2015
    • "Once the type of sample to be used has been selected, it is necessary to optimize the protocol for tissue disruption/cell-lysis or for the isolation of the proteins from body fluids using commercial reagents including protease and phosphatase inhibitors. When analysing serum/plasma, it may be necessary to use an albumin/IgG depletion kit to identify proteins whose concentration is low [15]. We have found that many proteins do not exist as a single form but as isoforms probably generated by post-translational modifications (PTM) of a given protein. "
    Full-text · Article · May 2015
    • "AB Sciex). The area under the most intense peak was calculated, and normalized to the internal standard (Takahashi and others 2013). The peak transition for the input internal standard was also used as a quality control measure. "
    [Show abstract] [Hide abstract] 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-PCR
    Full-text · Article · Jan 2015
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