Resolution and identification of major peanut allergens using a combination of fluorescence two-dimensional differential gel electrophoresis, Western blotting and Q-TOF mass spectrometry
Resolution and identification of major peanut allergens using a combination of fluorescence two-dimensional differential gel electrophoresis, Western blotting and Q-TOF mass spectrometry
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DOI：
10.1016/j.jprot.2009.02.002
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发表时间：
2009-04-13
影响因子：
3.3
通讯作者：
van Hengel, Arjon J.
中科院分区：
文献类型：
--
作者：
Chassaigne, Hubert;Tregoat, Virginie;van Hengel, Arjon J.
花生过敏是由几种称为过敏原的蛋白质引发的。本研究利用蛋白质组学工具对花生变应原的复杂性进行了研究。这项研究的优势在于结合了荧光二维差示凝胶电泳法的高分辨率和重复性，以及特异性免疫学检测和高分辨率质谱仪的多肽测序。花生蛋白与纯化的变应原(Ara h 1、Ara h 2或Ara h 3/4)在同一凝胶上进行差异标记，实现了花生蛋白在2D凝胶中的匹配。在一条约63-68 kDa的质量线上有10个蛋白质点可能对应于Ara-h1，在约16 kDa和18 kDa的两条不同质量线上的两对偶联与纯化的变应原Ara-h2相匹配。在质量约25 kDa和40-45 kDa处分别观察到Ara-h3/4的碱性和酸性亚基。用抗Ara h1、Ara h2和重组40 kDa的重组Ara h 3/4亚基的抗体(IgY)对花生变应原蛋白斑点的抗体结合能力进行了Western blotting检测。通过凝胶内消化和四极飞行时间质谱仪分析，最终确认了经2D电泳法匹配并经Western blotting鉴定的蛋白质斑点的同源性。利用我们建立的方法，建立了花生蛋白质二维图谱中两种不同亚型的变应原Arah1、变应原Arah2和六种变应原Arah3/4的定位和鉴定。(C)2009爱思唯尔B.V.保留所有权利。
Peanut allergy is triggered by several proteins known as allergens. In this study, the complexity the peanut allergome is investigated with proteomic tools. The strength of this investigation resides in combining the high-resolving power and reproducibility of fluorescence two-dimensional differential gel electrophoresis with specific immunological detection as well as polypeptide sequencing by high-resolution mass spectrometry. Matching of the peanut proteins in 2D gels was achieved by differential labelling whereby peanut proteins and purified allergens (Ara h 1, Ara h 2 or Ara h 3/4) were run on the same gel. Ten protein spots on a mass line of ca. 63-68 kDa were likely to correspond to Ara h 1. Two doublets on two different mass lines at ca. 16 and 18 kDa matched with purified allergen Ara h 2. The basic and acidic sub-units of Ara h 3/4 were observed at masses of ca. 25 kDa and 40-45 kDa, respectively. Subsequently the antibody-binding capacity of spots corresponding to peanut allergens was investigated by Western blotting of 2D gels using antibodies (IgY) raised against Ara h 1, Ara h 2 and the recombinant 40 kDa sub-unit of Ara h 3/4. Final confirmation of the identity of the protein spots matched after 2D electrophoresis and identified by Western blotting was obtained by in-gel digestion of protein spots and analysis by quadrupole time-of-flight mass spectrometry. By using the method developed in our work, the location and identification of two different isoforms of the allergen Ara h 1, the allergen Ara h 2 and six isoforms of the allergen Ara h 3/4 in 2D peanut protein maps was established. (c) 2009 Elsevier B.V. All rights reserved.