Proteomic analysis of putative latex allergens.
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.
Yagami
Takeshi
T
Division of Medical Devices, National Institute of Health Sciences, Kamiyoga 1-18-1, Setagaya-ku, Tokyo 158-8501, Japan. yagama@nihs.go.jp
Haishima
Yuji
Y
Tsuchiya
Toshie
T
Tomitaka-Yagami
Akiko
A
Kano
Hisao
H
Matsunaga
Kayoko
K
D000328:Adult; D000485:Allergens / Q000032:analysis* / Q000276:immunology; D030562:Databases, Protein; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D005260:Female; D006801:Humans; D007840:Latex / Q000737:chemistry* / Q000276:immunology; D020315:Latex Hypersensitivity / Q000276:immunology*; D008297:Male; D013058:Mass Spectrometry / Q000379:methods; D040901:Proteomics / Q000379:methods*
D000485:Allergens; D007840:Latex