Proteomic analysis of putative latex allergens

Takeshi Yagami, Yuji Haishima, Toshie Tsuchiya, Akiko Yagami, Hisao Kano, Kayoko Matsunaga

Research output: Contribution to journalArticle

43 Citations (Scopus)

Abstract

Background: Extensive analysis of allergenic proteins is generally time-consuming and labor-intensive. Accordingly, a rapid and easy procedure for allergen identification is required. As sequence information on proteins and genes is accumulated in databases, it is becoming easier to identify a candidate protein using proteomic strategies, i.e. two-dimensional gel electrophoresis, site-specific fragmentation, mass spectrometry and then database search. In this study, we evaluated the usefulness of a proteomic strategy for identifying putative allergens through its application to latex proteins. Methods: Latex proteins were separated with two-dimensional gel electrophoresis, and putative allergens were visualized by IgE immunoblotting using pooled serum from latex-sensitive patients. The IgE-interactive proteins were cut out from the negatively stained two-dimensional gel and subjected to in-gel digestion by trypsin. Then the resulting peptides were analyzed with mass spectrometry. Based on the mass spectrometric data we obtained, the allergen candidates were assigned by a database search. Results: Five previously reported allergens and five new allergen candidates were identified with the proteomic approach without isolating the individual proteins. Less than 1 mg of crude latex protein was sufficient for the entire protocol. Because plural proteins can be processed in parallel, analysis of about 50 IgE-interactive proteins was accomplished within 1 week. Conclusions: Analysis of putative allergens with proteomic strategies (allergenomics) is a promising avenue for rapid and exhaustive research. The high resolving power of two-dimensional gel electrophoresis is superior to conventional gel electrophoresis. Moreover, the notable sensitivity and speed of mass spectrometry have pronounced advantages over the N-terminal sequencing that has generally been used for protein identification.

Original languageEnglish
Pages (from-to)3-11
Number of pages9
JournalInternational Archives of Allergy and Immunology
Volume135
Issue number1
DOIs
Publication statusPublished - 07-10-2004

Fingerprint

Latex
Proteomics
Allergens
Proteins
Electrophoresis, Gel, Two-Dimensional
Immunoglobulin E
Mass Spectrometry
Gels
Databases
Immunoblotting
Trypsin
Electrophoresis
Digestion
Peptides

All Science Journal Classification (ASJC) codes

  • Immunology and Allergy
  • Immunology

Cite this

Yagami, Takeshi ; Haishima, Yuji ; Tsuchiya, Toshie ; Yagami, Akiko ; Kano, Hisao ; Matsunaga, Kayoko. / Proteomic analysis of putative latex allergens. In: International Archives of Allergy and Immunology. 2004 ; Vol. 135, No. 1. pp. 3-11.
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Yagami, T, Haishima, Y, Tsuchiya, T, Yagami, A, Kano, H & Matsunaga, K 2004, 'Proteomic analysis of putative latex allergens', International Archives of Allergy and Immunology, vol. 135, no. 1, pp. 3-11. https://doi.org/10.1159/000080036

Proteomic analysis of putative latex allergens. / Yagami, Takeshi; Haishima, Yuji; Tsuchiya, Toshie; Yagami, Akiko; Kano, Hisao; Matsunaga, Kayoko.

In: International Archives of Allergy and Immunology, Vol. 135, No. 1, 07.10.2004, p. 3-11.

Research output: Contribution to journalArticle

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AU - Yagami, Takeshi

AU - Haishima, Yuji

AU - Tsuchiya, Toshie

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AU - Matsunaga, Kayoko

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N2 - Background: Extensive analysis of allergenic proteins is generally time-consuming and labor-intensive. Accordingly, a rapid and easy procedure for allergen identification is required. As sequence information on proteins and genes is accumulated in databases, it is becoming easier to identify a candidate protein using proteomic strategies, i.e. two-dimensional gel electrophoresis, site-specific fragmentation, mass spectrometry and then database search. In this study, we evaluated the usefulness of a proteomic strategy for identifying putative allergens through its application to latex proteins. Methods: Latex proteins were separated with two-dimensional gel electrophoresis, and putative allergens were visualized by IgE immunoblotting using pooled serum from latex-sensitive patients. The IgE-interactive proteins were cut out from the negatively stained two-dimensional gel and subjected to in-gel digestion by trypsin. Then the resulting peptides were analyzed with mass spectrometry. Based on the mass spectrometric data we obtained, the allergen candidates were assigned by a database search. Results: Five previously reported allergens and five new allergen candidates were identified with the proteomic approach without isolating the individual proteins. Less than 1 mg of crude latex protein was sufficient for the entire protocol. Because plural proteins can be processed in parallel, analysis of about 50 IgE-interactive proteins was accomplished within 1 week. Conclusions: Analysis of putative allergens with proteomic strategies (allergenomics) is a promising avenue for rapid and exhaustive research. The high resolving power of two-dimensional gel electrophoresis is superior to conventional gel electrophoresis. Moreover, the notable sensitivity and speed of mass spectrometry have pronounced advantages over the N-terminal sequencing that has generally been used for protein identification.

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