Gas-phase Ni(II) affinities of alternative metal binding peptides from competitive threshold collision-induced dissociation

Abstract

The Ni(II) affinity of the polyhistidine tag is used in the purification of recombinant proteins by immobilized metal affinity chromatography. Here we measured the relative gas-phase Ni(II) affinities of four alternative metal binding (amb) peptides and the 7xHis tag using the competitive threshold collision-induced dissociation (TCID) technique. The general primary structure of the four amb peptides was acetyl-Aa1-Aa2-Gly3-Pro4-Aa5-Gly6-Cys7, designed to test whether the His1-Cys2 or Asp1-His2 would exhibit the higher Ni(II) affinity and whether the Tyr5 with its ability of forming long-range π-nickel interaction and hydrogen bonding would contribute to the Ni(II) affinity. The Cys7 is retained in all the amb sequences because previous research has shown that both the thiolate side group and carboxylate terminus simultaneously coordinate the metal ion. The Ni(II) affinity was measured using the dissociation of the [amb + Ni(II) + NTA]− complex, where NTA = nitrilotriacetic acid, which is a commonly used ligand for Ni(II) inside the IMAC column. The dissociation of [amb + Ni(II) + NTA]− produced two main product channels; [amb + Ni(II)]− + NTA and [NTA + Ni(II)]− + amb, whose competition was modeled by TCID to extract the relative gas-phase Ni(II) affinities. Extensive molecular modeling using PM6 located the low-energy structures whose molecular parameters were used in the TCID analyses if their collision cross sections agreed with those measured by traveling-wave ion mobility mass spectrometry and they were compatible with a concerted reaction. We compare the final results by conducting the TCID analyses using alternative PM6 parameters and by making the ternary complexes in acidic and basic solutions.