Morschheuser Lena, Mink Kathrin, Horst Ramona, Kallinich Constanze, Rohn Sascha
University of Hamburg, Hamburg School of Food Science, Institute of Food Chemistry, Grindelallee 117, D-20146 Hamburg, Germany.
University of Hamburg, Hamburg School of Food Science, Institute of Food Chemistry, Grindelallee 117, D-20146 Hamburg, Germany.
J Chromatogr A. 2017 Dec 1;1526:157-166. doi: 10.1016/j.chroma.2017.10.046. Epub 2017 Oct 20.
The chromatographic analysis of intact proteins is still challenging, especially when biological functions as antigenicity of proteins or peptides are in the focus. Traditional immunoassays provide information about the entirety of antigenic proteins/peptides, e.g., in ELISA assays. On the other hand, when focusing on the investigation of (cross) reactivity of antibodies, Western blot following gel-electrophoresis represents the method of choice. However, gel-electrophoresis is limited by the molecular weight and therefore, not suitable for peptides ≤3kDa or proteins ≥250kDa. Furthermore, for gaining detailed information about the protein sequence (e.g., via mass spectrometric analysis), a so called in-gel digest needs to be performed following electrophoretic separation and is therefore elaborate and accompanied by a significant loss of structural, and even more severe, conformational information. Here, protein analysis using HPTLC seems to be a promising alternative due to the high level of variability regarding the chromatographic system (multiple mobile and stationary phases, even mixed) and manifold detection as well as hyphenation possibilities. This study exemplarily focused on the immunological investigation of proteins in milk following thin-layer chromatographic separation. The detection of these antigens is mandatory, as they might trigger allergenic reactions in sensitized people. Besides the proof of its applicability on different stationary phase materials, the newly developed immunoassay can be used as an approach for semi-quantitative estimation of antigenic proteins. In addition to the analysis of intact food allergens, also analyzing peptides thereof is worth considering which can be realized using HPTLC-immunostaining as well.
完整蛋白质的色谱分析仍然具有挑战性，尤其是当蛋白质或肽的生物学功能如抗原性成为关注焦点时。传统免疫测定可提供有关抗原性蛋白质/肽整体的信息，例如在酶联免疫吸附测定（ELISA）中。另一方面，当专注于抗体（交叉）反应性的研究时，凝胶电泳后的蛋白质印迹法是首选方法。然而，凝胶电泳受分子量限制，因此不适用于分子量≤3 kDa的肽或≥250 kDa的蛋白质。此外，为了获得有关蛋白质序列的详细信息（例如通过质谱分析），需要在电泳分离后进行所谓的胶内消化，这一过程繁琐，且会导致大量结构信息甚至更严重的构象信息丢失。在此，由于色谱系统具有高度可变性（多种流动相和固定相，甚至混合相）、多种检测方式以及联用可能性，使用高效薄层色谱（HPTLC）进行蛋白质分析似乎是一种很有前景的替代方法。本研究以薄层色谱分离后牛奶中蛋白质的免疫学研究为例。检测这些抗原是必要的，因为它们可能会在过敏人群中引发过敏反应。除了证明其在不同固定相材料上的适用性外，新开发的免疫测定法还可作为一种对抗原性蛋白质进行半定量估计的方法。除了分析完整的食物过敏原外，分析其肽段也值得考虑，这也可以通过HPTLC免疫染色来实现。
