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The analog methanobactin (amb) peptide with the sequence ac‐His1‐Cys2‐Gly3‐Pro4‐Tyr5‐His6‐Cys7 (amb5A) will bind the metal ions of zinc, nickel, and copper. To further understand how amb5A binds these metals, we have undertaken a series of studies of structurally related heptapeptides where one or two of the potential His or Cys binding sites have been replaced by Gly, or the C‐terminus has been blocked by amidation. The studies were designed to compare how these metals bind to these sequences in different pH solutions of pH 4.2 to 10 and utilized native electrospray ionization (ESI) with ion mobility‐mass spectrometry (IM‐MS) which allows for the quantitative analysis of the charged species produced during the reactions. The native ESI conditions were chosen to conserve as much of the solution‐phase behavior of the amb peptides as possible and an analysis of how the IM‐MS results compare with the expected solution‐phase behavior is discussed. The oligopeptides studied here have applications for tag‐based protein purification methods, as therapeutics for diseases caused by elevated metal ion levels or as inhibitors for metal‐protein enzymes such as matrix metalloproteinases.

Electrospray ionization (ESI) can transfer an aqueous-phase peptide or peptide complex to the gas-phase while conserving its mass, overall charge, metal-binding interactions, and conformational shape. Coupling ESI with ion mobility-mass spectrometry (IM-MS) provides an instrumental technique that allows for simultaneous measurement of a peptide's mass-to-charge (m/z) and collision cross section (CCS) that relate to its stoichiometry, protonation state, and conformational shape. The overall charge of a peptide complex is controlled by the protonation of 1) the peptide's acidic and basic sites and 2) the oxidation state of the metal ion(s). Therefore, the overall charge state of a complex is a function of the pH of the solution that affects the peptides metal ion binding affinity. For ESI-IM-MS analyses, peptide and metal ions solutions are prepared from aqueous-only solutions, with the pH adjusted with dilute aqueous acetic acid or ammonium hydroxide. This allows for pH dependence and metal ion selectivity to be determined for a specific peptide. Furthermore, the m/z and CCS of a peptide complex can be used with B3LYP/LanL2DZ molecular modeling to discern binding sites of the metal ion coordination and tertiary structure of the complex. The results show how ESI-IM-MS can characterize the selective chelating performance of a set of alternative methanobactin peptides and compare them to the copper-binding peptide methanobactin. Video Link The video component of this article can be found at https://www.jove.com/video/60102/ 19. Recently, a series of alternative methanobactin (amb) oligopeptides with the 2His-2Cys binding motif have been studied 20,21 , and their Zn(II) and Cu(I/II) binding properties characterized. Their primary amino acid

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