The characterization of cell surface receptor complexes by affinity chromatography, liquid chromatography and tandem mass spectrometry
thesisposted on 23.05.2021, 10:06 by Jaimie Dufresne
Cell surface receptors are of critical importance to the treatment of disease but are difficult to isolate and identify by classical approaches. Here, a robust and general method for capturing a receptor complex from the surface of live cells with ligands presented on nanoscopic beads is demonstrated. Two forms of affinity chromatography: the presentation of a biotinylated ligand to the surface of live cells and recovered by classical affinity chromatography was compared to the presentation of the ligand on the surface of nanoscopic chromatography beads for the isolation of the IgG-FcR complex from the surface of live cells. The IgG ligand was first characterized by LC-ESI-MS/MS and compared to controls by classical statistical methods in order to minimize the total error rate of protein identification. The first strategy was to isolate the IgG-FcR complex using biotinylated IgG (IgG-B) compared to IgG with a DTT-cleavable biotin spacer arm (IgG-SS-B) to activate and capture the receptor associated supramolecular complex. In this method, live cells were incubated on ice with IgG, IgG-B or IgG-S-S-B, for 30 minutes, washed and disrupted by French press. After washing, the IgG-B and IgG-S-S-B ligands were eluted from a streptavidin-agarose column in DTT followed by mercaptoethanol, 2M NH4OH and then 50mM biotin in 2 M NH4OH and the ligand remaining on the columns was scrubbed with 2% SDS or digested with trypsin. The analysis of the eluted fractions and SDS scrub by dot blots or Western blots with streptavidin-HRP (SA-HRP) or donkey anti human IgG-HRP (Dk anti-hIgG-HRP) probes agreed with liquid chromatography and tandem mass spectrometry (LC-ESI-MS/MS) of the tryptic peptides that IgG-B or IgG-S-S-B was non-specifically eluted by ammonium hydroxide buffer. LC-ESI-MS/MS of the DTT eluted fraction from the charged streptavidin column (agarose-SA-B-S-S-IgG) to release the S-IgG ligand showed detectable amounts of the S-IgG complex and minimal amounts of Fc and Fcrl proteins. Some of the known components of the Fc mediated phagocytic pathway were enriched in DDT and BME in the Biotin-S-S-IgG-FCGR compared to Biotin-IgG controls. Many variations of the classical ligand affinity chromatography method for the isolation of cell surface receptors were investigated including the use of detergents, heat aggregated ligands, the use of cross linkers and the capture of IgG over streptavidin or protein G chromatography. None of these methods specifically captured Fc receptors however the identified proteins were different from background binding controls. In the second strategy, IgG coated PMMA and melamine micro and glass nano beads were presented to live cells, homogenized by French pressing, isolated by ultracentrifugation and then extracted by a salt and acetonitrile step gradient. Laser confocal scanning microscopy, dot blots with Coomassie staining or Western blot all confirm the three nano and micro bead surfaces: silica, melamine and PMMA were coated in IgG and were able to bind RAW 264.7 macrophages. Fc receptors were identified in significant numbers in the silica nano treatments compared to controls as well as members of the FcR mediated phagocytic pathway with very little background binding. In contrast to the strategy of classical affinity chromatography applied to integral membrane receptors, the use of ligand coated micro, and as shown here for the first time, silica nano beads, was effective at binding and capturing a receptor from the surface of a live cell and may serve as a general method for isolating receptor complexes on the nano scale.