New Insights into the DT40 B Cell Receptor Cluster Using a Proteomic Proximity Labeling Assay

Abstract

Background: B cell receptor (BCR) clusters modulate BCR signaling in B-lymphocytes.Results: We used a quantitative proteomic proximity assay to analyze the BCR cluster in DT40 cells.Conclusion: Our proximity labeling assay identified novel components of the BCR cluster linked to integrin signaling.Significance: We provide new insights into BCR assembly and identify new and unexpected targets for further functional analysis.

Filling the Void: Proximity-Based Labeling of Proteins in Living Cells

“…Another HRP-based approach called s elective p roteomic p roximity l abeling a ssay using t yramide (SPPLAT) [37, 38] utilizes HRP conjugated antibodies or ligands with biotin tyramide compounds and H 2 O 2 to biotinylate proximate proteins on the cell surface. The biotin-tyramide derivatives have a cleavable linker that facilitates access of streptavidin to biotinylated proteins, recovery of biotinylated proteins from streptavidin, and reduced membrane permeability [37, 38].…”

“…The biotin-tyramide derivatives have a cleavable linker that facilitates access of streptavidin to biotinylated proteins, recovery of biotinylated proteins from streptavidin, and reduced membrane permeability [37, 38]. Used in combination with stable isotope labeling for quantitative proteomics in a lymphocyte cell line, SPPLAT was applied to study the composition of B-cell receptor (BCR) cluster with an HRP-conjugated anti-IgM antibody.…”

“…One limitation observed with HRP-based methods has been membrane permeability of some labeling reagents that may potentially lead to false positives [29, 37]. It is difficult to distinguish a clear advantage between EMARS or SPPLAT, except for the difference in labeling reagents.…”

Multidimensional proteomics for cell biology

“…The proximity ligation approach has also been extended to study plasma membrane proteins exposed to the extracellular milieu. This is carried out by incubating intact cells with antibody-peroxidase conjugates that will specifically interact with a plasma membrane protein for the subsequent conjugation of biotin-tyramide molecules to proximal proteins 69 . Nonetheless, such methods involve the targeted expression of the recombinant ligase and are therefore more suited to application in cell lines rather than for primary cells and tissues because of the relative ease of transfecting or infecting cell lines.…”

Protein networks and activation of lymphocytes

“…A collection of approaches to define PPIs using AP-MS has been reviewed elsewhere [29–36]. Many of these approaches have been implemented to interrogate PPI networks in lymphocyte signaling [37–40]. Recently, mice bearing a genetic tag that permit AP-MS of signaling complexes containing ZAP-70, LAT, and SLP-76 was described using label-free quantitation.…”