Method for B Cell Receptor Enrichment in Malignant B Cells

Author:

Bhattacharyya Puja12,Christopherson Richard I.3,Skarratt Kristen K.14ORCID,Fuller Stephen J.14ORCID

Affiliation:

1. Sydney Medical School Nepean, Faculty of Medicine and Health, The University of Sydney, Penrith, NSW 2750, Australia

2. Blacktown Hospital, Blacktown Rd., Blacktown, NSW 2148, Australia

3. School of Life and Environmental Sciences, University of Sydney, Sydney, NSW 2006, Australia

4. Nepean Hospital, Derby Str., Kingswood, NSW 2747, Australia

Abstract

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.

Funder

Sydney West Translational Cancer Research Centre, the Cancer Institute of NSW

Gary and Marion Krelle and the Nepean Medical Research Fund

Publisher

MDPI AG

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