Abstract
Type 1 diabetes (T1D) is expected to cause significant changes in the serum proteome; however, few studies have systematically assessed the proteomic profile change associated with the disease. In this study, a semiquantitative spectral counting-based two dimensional liquid chromatography mass spectrometry platform was used to analyze serum samples from T1D patients and controls. In this discovery phase, significant differences were found for 21 serum proteins implicated in inflammation, oxidation, metabolic regulation, and autoimmunity. To assess the validity of these findings, six candidate proteins including adiponectin, insulin-like growth factor binding protein 2, serum amyloid protein A, C-reactive protein, myeloperoxidase, and transforming growth factor beta induced were selected for subsequent immune assays for 1139 T1D patients and 848 controls. A series of statistical analyses using cases and controls matched for age, sex, and genetic risk confirmed that T1D patients have significantly higher serum levels for four of the six proteins: adiponectin (odds ratio (OR) = 1.95, p = 10(-27)), insulin-like growth factor binding protein 2 (OR = 2.02, p < 10(-20)), C-reactive protein (OR = 1.13, p = 0.007), serum amyloid protein A (OR = 1.51, p < 10(-16)); whereas the serum levels were significantly lower in patients than controls for the two other proteins: transforming growth factor beta induced (OR = 0.74, p < 10(-5)) and myeloperoxidase (OR = 0.51, p < 10(-41)). Compared with subjects in the bottom quartile, subjects in the top quartile for adiponectin (OR = 6.29, p < 10(-37)), insulin-like growth factor binding protein 2 (OR = 7.95, p < 10(-46)), C-reactive protein (OR = 1.38, p = 0.025), serum amyloid protein A (OR = 3.36, p < 10(-16)) had the highest risk of T1D, whereas subjects in the top quartile of transforming growth factor beta induced (OR = 0.41, p < 10(-11)) and myeloperoxidase (OR = 0.10, p < 10(-43)) had the lowest risk of T1D. These findings provided valuable information on the proteomic changes in the sera of T1D patients.
Highlights
From the ‡Center for Biotechnology and Genomic Medicine; §Department of Pathology, Georgia Health Sciences University, Augusta, GA; ¶Atlanta Diabetes Associates, Atlanta, GA; ʈPediatric Endocrine Associates, Atlanta, GA; **Southeastern Endocrine and Diabetes, Atlanta, GA
To discover and validate serum proteomic differences between Type 1 diabetes (T1D) and AbN control groups, we designed our experiments comprising of a discovery phase and a validation phase (Fig. 2)
We found that all six molecules were significantly associated with increased or decreased risk of having T1D: ADIPOQ (OR ϭ 1.95), insulin-like growth factor binding protein 2 (IGFBP2) (OR ϭ 2.02), C-reactive protein (CRP) (OR ϭ 1.13), serum amyloid protein A (SAA) (OR ϭ 1.51), transforming growth factor beta induced (TGFBI) (OR ϭ 0.74), and MPO (OR ϭ 0.51)
Summary
T1D, Type 1 diabetes; 2D, Two dimensional; AbN, Autoantibody-negative; ADIPOQ, Adiponectin; CI, Confidence intervals; CRP, C-reactive protein; CV, Coefficient of variation; EAE, Encephalomyelitis; ESI, Electrospray ionization; FDR, First-degree relatives; GP, General population; IFN-␥, Interferon gamma; IGF, Insulin-like growth factor; IGFBP2, Insulin-like growth factor binding protein 2; IL-12, Interleukin-12; IL-1, Interleukin 1 beta; IL-6, Interleukin-6; IL-8, Interleukin-8; IL-12, Interleukin-12; LTQ, Linear ion-trap mass spectrometry; MFI, Median fluorescence intensity; MPO, Myeloperoxidase; MS, Mass spectrometry; PANDA, Prospective Assessment in Newborns of Diabetes Autoimmunity; OR, Odds ratio; RP, Reverse phase; SAA, Serum amyloid A; SCX, Strong cation exchange; T2D, Type 2 diabetes; TGFBI, Transforming growth factor beta induced; TGF-, Transforming growth factor beta; TNF-␣, Tumor necrosis factor-alpha. Serum Protein Profiles in Type 1 Diabetes Patients large variation between individuals and relatively small differences between study groups. Spectral counting is a simple, low-cost and semiquantitative approach to assess MS-based protein differences.[8] This approach was used in this study to compare serum protein differences between T1D patients and autoantibody-negative (AbN) control subjects. Irrespective of the technical platforms used in the discovery phase, the selected biomarker candidates have to be validated in subsequent studies. Our validation phase included six candidate proteins that showed significant differences in the discovery phase: adiponectin (ADIPOQ), insulin-like growth factor binding protein 2 (IGFBP2), serum amyloid protein A (SAA), C-reactive protein (CRP), myeloperoxidase (MPO), and transforming growth factor beta induced (TGFBI). This study provided convincing evidence that the six proteins were significantly altered in T1D patients
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