Exploiting His-Tags for Absolute Quantitation of Exogenous Recombinant Proteins in Biological Matrices: Ruthenium as...

Metal labeling and ICP MS detection offer an alternative to commonly accepted techniques that are currently used to quantitate exogenous proteins in vivo, but modifying the protein surface with metal-containing groups inevitably changes its biophysical properties and is likely to affect trafficking and biodistribution. The approach explored in this work takes advantage of the presence of hexa-histidine tags in many recombinant proteins, which have high affinity toward a range of metals. While many divalent metals bind to poly histidine sequences reversibly, oxidation of imidazole-bound Co^II or Ru^II is known to result in a dramatic increase of the binding strength. In order to evaluate the feasibility of using imidazole-bound metal oxidation as a means of attaching permanent tags to polyhistidine segments, a synthetic peptide YPDFEDYWMKHHHHHH was used as a model. Ru^II can be oxidized under ambient (aerobic) conditions, allowing any oxidation damage to the peptide beyond the metal-binding site to be avoided. The resulting peptide-Ru^III complex is very stable, with the single hexa-histidine segment capable of accommodating up to three metal ions. Localization of Ru^III within the hexa-histidine segment of the peptide was confirmed by tandem mass spectrometry. The Ru^III/peptide binding appears to be irreversible, with both low- and high-molecular weight biologically relevant scavengers failing to strip the metal from the peptide. Application of this protocol to labeling a recombinant form of an 80 kDa protein transferrin allowed Ru^III to be selectively placed within the His-tag segment. The metal label remained stable in the presence of ubiquitous scavengers and did not interfere with the receptor binding, while allowing the protein to be readily detected in serum at sub-nM concentrations. The results of this work suggest that ruthenium lends itself as an ideal metal tag for selective labeling of His-tag containing recombinant proteins to enable their sensitive detection and quantitation with ICP MS.