(215b) An Orthogonal Affinity Capture and Detection Strategy for Identifying and Characterizing Phosphoproteins Directly from Sds-Polyacrylamide Gels: Inorganic Chemistry to the Rescue!

An antibody-free process workflow is described involving orthogonal phosphomonoester-selective binding strategies. First, complex protein samples are enriched for phosphoproteins using an alkoxide-bridged dinuclear zinc complex conjugated to agarose beads under nondenaturing conditions, at neutral pH. The enriched phosphoproteins are separated by conventional SDS-polyacrylamide gel electrophoresis and a fluorophore-conjugated alkoxide-bridged dinuclear zinc complex is employed to selectively highlight phosphoproteins via binding to the phosphomonoester dianion moieties of serine, threonine, and tyrosine. Interaction with other anionic residues, including carboxylate residues on proteins, is insignificant. As little as 1 ng of phosphoprotein is detectable by this method using standard charge-coupled device camera- or laser scanner-based imaging systems. Then, phosphoprotein bands are excised from the gel, subjected to proteolytic digestion and constituent phosphopeptides subsequently purified using Titania thin-film coated magnetic beads at acidic pH. Phosphopeptides are eluted at alkaline pH and directly characterized by MALDI-TOF or tandem mass spectrometry. Phosphopeptides can readily be identified with minimal contamination of samples by acidic peptides, as is often encountered using conventional trivalent ferric- or gallium-based immobilized metal ion affinity chromatography (IMAC) approaches. The workflow does not require two-dimensional gel electrophoresis or nano-liquid chromatography, making analysis of phosphoproteins accessible to a broad spectrum of Life Sciences investigators.