B cells are central to the adaptive immune response and provide long-lasting immunity after infection. B cell activation is mediated by the surface membrane-bound B cell receptor (BCR) following recognition of a specific antigen. The BCR has been challenging to analyse using mass spectrometry (MS) due to the difficulty of isolating and enriching this membrane-bound protein complex. There are approximately 120,000 BCRs on the B cell surface; however, depending on the B cell activation state, there may be hundreds-of-millions to billions of proteins in a B cell. Consequently, advanced proteomic techniques such as MS workflows that use purified proteins to yield structural and protein-interaction information have not been published for the BCR complex. This paper describes a method for enriching the BCR complex that is MS-compatible. The method involves a Protein G pull down on agarose beads using an intermediary antibody to each of the BCR complex subcomponents (CD79a, CD79b, and membrane immunoglobulin). The enrichment process is shown to pull down the entire BCR complex and has the advantage of being readily compatible with further proteomic study including MS analysis. Using intermediary antibodies has the potential to enrich all isotypes of the BCR, unlike previous methods described in the literature that use protein G-coated beads to directly pull down the membrane IgG (mIgG) but cannot be used for other mIg isotypes.
B细胞是适应性免疫应答的核心，在感染后提供持久免疫力。B细胞活化由表面膜结合型B细胞受体（BCR）在识别特异性抗原后介导。由于该膜结合蛋白复合物的分离与富集难度较高，使用质谱（MS）分析BCR一直面临挑战。B细胞表面约有12万个BCR；然而根据B细胞活化状态，单个B细胞内可能包含数亿至数十亿个蛋白质。因此，目前尚未见采用纯化蛋白质获取结构和蛋白质相互作用信息的质谱工作流程等先进蛋白质组学技术应用于BCR复合物的报道。本文描述了一种与质谱兼容的BCR复合物富集方法。该方法通过针对BCR复合物各亚组分（CD79a、CD79b和膜免疫球蛋白）的中介抗体，在琼脂糖珠上进行Protein G下拉实验。该富集过程可捕获完整的BCR复合物，并具备易于兼容包括质谱分析在内的进一步蛋白质组学研究的优势。相较于文献中既往采用Protein G包被磁珠直接下拉膜结合型IgG（mIgG）但无法用于其他膜结合型免疫球蛋白同种型的方法，使用中介抗体有望富集所有同种型的BCR。
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