J Korean Med Sci.  2005 Aug;20(4):579-585. 10.3346/jkms.2005.20.4.579.

Proteomic Analysis of Differently Expressed Proteins in a Mouse Model for Allergic Asthma

• Jeong HS
• Rhim TY
• Ahn MH
• Yoon PO
• Kim SH
• Chung IY
• Uh ST
• Kim SI
• Park CS

• KMID: 1712736
• DOI: http://doi.org/10.3346/jkms.2005.20.4.579

Abstract

Allergic asthma is associated with persistent functional and structural changes in the airways and involves many different cell types. Many proteins involved in allergic asthma have been identified individually, but complete protein profiles (proteome) have not yet been reported. Here we have used a differential proteome mapping strategy to identify tissue proteins that are differentially expressed in mice with allergic asthma and in normal mice. Mouse lung tissue proteins were separated using two-dimensional gel electrophoresis over a pH range between 4 and 7, digested, and then analyzed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MS). The proteins were identified using automated MS data acquisition. The resulting data were searched against a protein database using an internal Mascot search routine. This approach identified 15 proteins that were differentially expressed in the lungs of mice with allergic asthma and normal mice. All 15 proteins were identified by MS, and 9 could be linked to asthma-related symptoms, oxidation, or tissue remodeling. Our data suggest that these proteins may prove useful as surrogate biomarkers for quantitatively monitoring disease state progression or response to therapy.

Keyword

Electrophoresis; Gel; Two-Dimensional; Disease Models; Animal; Asthma; Proteomics; MALDITOF/MS; Chi313 protein; mouse

MeSH Terms

Animals
Asthma/genetics/immunology/*metabolism
Comparative Study
Disease Models, Animal
Electrophoresis, Gel, Two-Dimensional
Gene Expression/immunology
Gene Expression Profiling
Lung/immunology/metabolism/pathology
Male
Mice
Mice, Inbred BALB C
Ovalbumin/immunology
Proteome/*analysis/genetics/immunology
Proteomics/methods
RNA, Messenger/genetics/metabolism
Research Support, Non-U.S. Gov't
Reverse Transcriptase Polymerase Chain Reaction
Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

Figure

• 

  Fig. 1 Construction of the mouse model for allergic asthma. (A) Schematic representation of ovalbumin (OVA) sensitization and challenge protocol. (B) Airway responsiveness to methacholine in OVA-sensitized mice at 24 hr after the last challenge with saline (diamonds) or OVA (black rectangles). The results are expressed as the arithmetic means±SEM (n=6 per group). Significant differences from saline-challenged animals: *p<0.05; †p<0.01. (C) Numbers of mononuclear cells recovered in bronchial alveolar (BAL) fluid after saline or OVA challenge. Differential cell counts were performed on a minimum of 500 cells per sample to identify eosinophils (black), neutrophils (open), macrophages (hatched), and lymphocytes (gray). Challenge with OVA significantly increased the total cell numbers. Eosinophils and macrophages recovered in BAL fluid were significantly increased, whereas the neutrophil and lymphocyte counts were unchanged in OVA-challenged mice.

• 

  Fig. 2 2-dimension electrophoresis (DE) of proteins from normal (Sham) and allergic asthma (OVA) mouse lung tissues. Proteins from the whole lung were extracted and separated on an IPG strip with a nonlinear gradient of pH 3-10, followed by separation in the second dimension on an 8-18% SDS polyacrylamide gel. The gel was stained with Coomassie Blue G-250. Spots 1 was determined to be transthyretin.

• 

  Fig. 3 2-DE of proteins from normal (Sham) and allergic asthma (OVA) mouse lung tissues. In the first dimension, proteins were loaded on a 24-cm IPG strip with a linear gradient of pH 4-7. A 12% SDS polyacrylamide gel was used for second dimension separation. Proteins were visualized by silver staining. Indicated spots represent proteins differentially expressed between normal and asthmatic lung tissues.

• 

  Fig. 4 Protein expression levels were determined by relative intensity using image analysis. Normalized spot intensities of the asthma versus normal control (sham) group were compared. Mean intensity and spot intensities on individual gels are shown. *p<0.05; †p<0.01, ‡p<0.001 by Student's t-test.

• 

  Fig. 5 RT-PCR analysis of the mRNA expression of differentially expressed YM proteins. Primers specific for YM1 and YM2 were used as indicated to amplify the transcripts from total RNA isolated from normal and asthmatic lung tissues. RT-PCR of the housekeeping gene GAPDH was used to control for RNA variation (bottom).

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