REGULAR ARTICLE Sub-proteome analysis of novel IgE-binding proteins from Bermuda grass pollen Shao-Hsuan Kao 1 , Song-Nan Su 2 , Shih-Wen Huang 3 , Jaw-Ji Tsai 4 and Lu-Ping Chow 1, 5 1 Institute of Biochemistry and Molecular Biology, College of Medicine, National Taiwan University, Taipei, Taiwan 2 Department of Medical Researchand Education, Veterans General Hospital-Taipei, Taiwan 3 Department of Pediatrics, Division of Immunology and Allergy, University of Florida, Gainesville, FL, USA 4 Section of Allergy and Immunology, CathayGeneral Hospital, Taipei, Taiwan 5 Department of Medical Genetics, National Taiwan University Hospital, Taipei, Taiwan Bermuda grass (Cynodon dactylon) pollen (BGP) is one of the most common causes of airway allergic disease, and has been shown to contain over 12 allergenic proteins on 1-D immunoglo- bulin E (IgE) immunoblots. However, only a few allergens have been identified and character- ized. Cyn d 1 is a major allergen and the most abundant protein in BGP, representing 15% of the whole-pollen extract. To investigate variability in the IgE-reactive patterns of BGP-sensitized patients and to identify other prevalent allergens, a BGP extract was passed through an affinity column to remove Cyn d 1, and the non-bound material was collected and analyzed by 2-DE. IgE- reactive proteins were subsequently characterized by immunoblotting using serum samples from ten BGP-allergic patients. The prevalent IgE-reactive proteins were identified by MALDI- TOF MS, N-terminal sequence similarity, and LC-MS/MS. Here, we present a sub-proteome approach for allergen investigation and its use for determining BGP 2-DE profiles and identify- ing six novel allergens. Received: September 10, 2004 Revised: November 18, 2004 Accepted: December 27, 2004 Keywords: Allergen / Bermuda grass pollen / Immunoglobulin E-reactive protein / Protome analysis Proteomics 2005, 5, 3805–3813 3805 1 Introduction Grass pollen allergens trigger immunoglobulin E (IgE)- mediated type I allergic diseases, such as allergic rhinitis, conjunctivitis, and bronchial asthma [1–3]. Bermuda grass (Cynodon dactylon) pollen (BGP) is one of the causative agents of asthma [4–6]. It is endemic in warm temperate and subtropical climates, such as the southwestern United States, northern and central Australia, South Africa, and Taiwan. In Taiwan, approximate 27% of asthmatic patients have positive immediate skin hypersensitive responses to the crude pollen extract [7]. A PBS crude extract of BGP contains a very complex mixture of IgG- and IgE-reactive components [8], but only a few allergens (Cyn d 1, Cyn d 7, Cyn d 12, BG60, and Cyn d Bd46K) have been identified [9–13]. The major allergen, Cyn d 1, is the most abundant protein (15% of the total crude extract) in BGP and gives a 76–100% posi- tive reaction rate with IgE in the sera of patients allergic to BGP [14–16]. Cyn d 7 is a calcium-binding protein, and its allergenicity and cross-reactivity have been investigated in other pollens, such as rape [17] and birch [18]. Cyn d 12 is a member of the profilins, which have been described as pan- allergens responsible for about 20% of the cross-reactivity seen in patients with pollen and food allergies [19]. Two other Correspondence: Professor Lu-Ping Chow, Institute of Biochem- istry and Molecular Biology, College of Medicine, National Tai- wan University, No. 1, Sec. 1, Jen-Ai Rd, Taipei, Taiwan 106 E-mail: lupin@ha.mc.ntu,edu.tw Fax: 1886-2-23958814 Abbreviations: BGP , Bermuda grass pollen; IgE, immunoglobu- lin E; dC1BCE, Cyn d 1-depleted BGP crude extract; EF-2, elonga- tion factor 2 ; MDH, malate dehydrogenase; PR, pathogenesis- related protein  2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.de DOI 10.1002/pmic.200401229