Proteomic analysis of ovomucoid hypersensitivity in mice by two-dimensional difference gel electrophoresis (2D-DIGE)

Abstract

There is a need to develop reliable methods to assess the safety of genetically modified and other novel foods. The aim of this study was to identify protein biomarkers of food allergy in mice exposed to ovomucoid (OVM), a major food allergen found in chicken egg white. BALB/c mice were repeatedly sensitized by gavage with OVM and cholera toxin (CT) and control mice were exposed to a mixture of amino acids with CT. At the endpoint, all mice were challenged intraperitoneally with OVM and alum. Type-1 hypersensitivity was confirmed in OVM-sensitized mice by observation of clinical signs of anaphylaxis and elevated levels of plasma histamine, OVM-specific IgE and OVM-specific IgG by ELISA. Differential protein expression was assessed in albumin-depleted plasma as well as in mesenteric lymph node, liver, spleen, and ileum by two-dimensional difference gel electrophoresis (2D-DIGE). Differentially expressed proteins were identified by liquid chromatography with tandem mass spectrometry. Plasma proteins overexpressed in OVM-sensitized mice included haptoglobin (41-fold), serum amyloid A (19-fold) and peroxiredoxin-2 (1.9-fold). Further validation of these plasma proteins in other animal models of food allergy with different food allergens is required to assess their potential as candidate biomarkers for use in evaluating the allergenicity of novel foods.

Fig. 1

Histamine, OVM-specific IgE and IgG are elevated in OVM-challenged mice. (a) Histamine; (b) OVM-specific IgE; and (c) OVM-specific IgG, were measured in pooled plasma samples (n = 5 pools with 3 mice/pool) of amino acid control mice and OVM-sensitized mice. Plasma was collected 40 min following IP challenge with OVM and assayed by ELISA. Results represent the mean ± SEM. *Significantly different from control (p ⩽ 0.05).

Fig. 2

2D-DIGE overlay image of differentially expressed plasma proteins in OVM-challenged mice. 2D-DIGE overlay image of protein spots comparing albumin-depleted plasma from unsensitized control mice labeled with Cy3 (green) to OVM-sensitized mice labeled with Cy5 (red), and an internal standard sample common to all gels labeled with Cy2 (blue). Proteins with similar expression levels in control and OVM-sensitized mice appear white in color. Plasma was collected 40 min following intraperitoneal challenge with ovomucoid. This image is representative of one of four gels analyzed.

Fig. 3

2D-DIGE gray scale image of plasma protein expression in control and OVM-challenged mice. Analytical 2D-DIGE gray scale images of samples depicted in Fig. 2 used to quantify changes in protein spot abundance in albumin-depleted plasma from (a) unsensitized control mice labeled with Cy3 and (b) OVM-sensitized mice labeled with Cy5, electrophoresed within the same gel. Protein spots that were significantly increased (change ⩾ 1.9-fold, p ⩽ 0.06) in OVM-sensitized mouse plasma are indicated. These images of individually labeled samples are representative of one the four gels analyzed.