Application of Novel Proteomic Methods to B Cells and the B Cell Receptor

Abstract

B cells are an integral part of the adaptive immune response with expression of surface immunoglobulin in a B cell receptor (BCR) complex. BCR engagement with antigen results in activation of B cells to eliminate targeted foreign pathogens. Subversion of normal cellular development in B cell neoplasms is mediated through transmembrane receptors including the BCR. There is a need to refine proteomic methods to capture dynamic interactions that occur between the native BCR and other transmembrane proteins. Cross-linking Mass spectrometry (XL-MS) has made significant contributions to studying protein-protein interaction (PPIs). However, XL-MS has not been widely used to study B cells, particularly membrane bound surface receptors. In this thesis, the optimal XL-MS conditions were determined using the membrane fraction (MF) of the Raji cell line. It was established that 5 mM disuccinimidyl sulfoxide added to a 1-2 μg/μL protein density cell suspension post soft lysis at room temperature for 30 minutes was optimal for XL-MS of the malignant B cell MF. The optimised XL-MS technique was applied to both Raji (High Grade) and OSU-CLL (low grade) malignant cell lines with the BCR sequentially in resting, activated and inhibited states to determine differences in PPIs. The PPI maps generated identified several clinically relevant proteins including prohibitin and alpha-enolase. To improve capture of the relatively low abundant BCR in our XL-MS dataset, a targeted purification using anti-BCR antibody with beads was developed. The most effective BCR enrichment occurred with the anti-CD 79a pull down to 500 μg of protein with stringent washing steps and sequencing the incubation of the beads with the antibody first followed by addition of the membrane fraction. Preliminary studies using XL-MS to analyse the membrane fraction of Raji cells combined with BCR enrichment was promising but further refinement of the technique is required to identify crosslinking partners of the BCR.