Differential proteome analysis of serum proteins associated with the development of type 2 diabetes mellitus in the KK-A(y) mouse model using the iTRAQ technique.

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.