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

Abstract

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 imidazolebound 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 metalbinding 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.

Ruthenium coordination preferences in imidazole‐containing systems revealed by electrospray ionization mass spectrometry and molecular modeling: Possible cues for the surprising stability of the Ru (III)/tris (hydroxymethyl)‐aminomethane/imidazole complexes

“…Despite an impressive variety of various ruthenium (III) complexes observed in the ESI mass spectra acquired in aqueous solution in the presence of tris (hydroxymethyl)‐aminomethane, it is worth noting that most of these complexes have a common structural feature, ie, association of two tris (hydroxymethyl)‐aminomethane molecules with a single‐metal atom. Introduction of imidazole into solution produces a remarkable effect of eliminating the structural variety, with the mass spectrum displaying a single abundant metal‐containing ion, [Ru III + 2 T + 2Im(C 3 N 2 H 4 ) – 2H] + . Nevertheless, the Ru III T 2 core (common to most complexes observed prior to the imidazole addition) appears to be preserved.…”

“…The reactivity of commonly used buffering compounds towards a wide range of biomolecules, particularly ones incorporating metal ions, is now widely recognized . Tris (tris (hydroxymethyl)‐aminomethane) in particular is known to form complexes with a variety of transition metals, and we recently observed facile oxidation and ligand exchange within the [Ru II (NH 3 ) 5 Cl] + complex upon its placement in Tris buffer under aerobic conditions . Earlier studies of Ru III complexation by tris (hydroxymethyl)‐aminomethane resulted in the complex structure assignment as [(RuTCl) 2 ]Cl 2 , where T denotes tris (hydroxymethyl)‐aminomethane .…”

“…Not only would it have a negative effect on the thermodynamic stability of the complexes but may also impact the kinetics of the assembly process, providing an opportunity for other, less stable complexes to be formed and persist should they behave as kinetic traps. This is a serious concern, as such kinetic traps (if they exist) may also affect ruthenium association with hexa‐histidine segments of recombinant proteins . Interaction of the ruthenium/tris (hydroxymethyl)‐aminomethane complexes with hexa‐histidine segments was evaluated in this work by using a synthetic hexapeptide HHHHHH.…”

“…However, introduction of a single cysteine residue into engineered‐in segments containing the hexa‐histidine sequence was shown to improve the efficiency of recombinant protein labeling with both Tc and Re, indicating that at least for these two metals, imidazoles can be outcompeted by thiols (thiolates) as optimal ligands. Recently, we explored the utility of His‐tags for site‐specific protein labeling with several less commonly used transition metals and found that facile formation of the exchange‐inert hexa‐histidine complexes with Ru III occurs when the latter is introduced in the form of a tris (hydroxymethyl)aminomethane (Tris base, T) complex . The ease of preparation and the remarkable stability of the Ru III /hexa‐histidine complexes are exciting, as they suggest that this metal can be used for the site‐specific labeling of recombinant proteins without the need to introduce highly reactive amino acids (such as unpaired cysteines) in the His‐tags, thereby enabling efficient labeling without compromising protein stability.…”

“…One remarkable feature of the ruthenium/imidazole (Im) complexes observed in our previous work was that two T ligands remained bound to the metal upon its association with either free imidazoles or a hexa‐histidine segment of a model peptide, reminiscent of the preservation of the metal/tricarbonyl core upon Tc or Re association with the hexa‐histidine/thiol systems . Despite the extensive body of knowledge accumulated over the past several decades vis‐à‐vis ruthenium reactivity towards a wide range of compounds, surprisingly little is known about this metal's behavior upon complexing with Im and T ligands (in fact, their combination had never been investigated vis‐à‐vis ruthenium complexation).…”