Ion Mobility-Mass Spectrometry Techniques for Determining the Structure and Mechanisms of Metal Ion Recognition and Redox Activity of Metal Binding Oligopeptides

Abstract

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

Formation of Co(II), Ni(II), Zn(II) complexes of alternative metal binding heptapeptides and nitrilotriacetic acid: Discovering new potential affinity tags

“…Our previous ESI-IM-MS amb analyses, have shown that the negatively-charged complexes are of particular interest because they correlate to the charge states that relate to the protonation state of the Cys, His, or carboxyl termini which are the potential metal ligand sites and they exhibit a distinct pH-dependence. [8][9][10][11][12][13][14][15] In Figure 3 we show the identity and the relative percentage of these negatively charged complexes for amb 5 G to R. Only G primarily formed the [amb 5 -6H+2Ni(II)] 2complex that related to six negatively-charged sites on the amb 5G peptide. Species G, as in the Co(II) analyses, also formed the negatively charged dimer binding 2Ni(II), [diamb 5ox -6H+2Ni(II)] 2-, where the 2Cys oxidized to form a disulfide bond.…”

“…Previous electrospray -ion mobility -mass spectrometry (ESI-IM-MS) studies of methanobactin 6,7 from Methylosinus trichosporium (mb-OB3b) and the amb peptides whose primary structure replaced the two enethiol oxazolone of mb-OB3b with the 2His-2Cys substituent groups of a zinc finger protein, have shown their potential for applications as affinity tags. [8][9][10][11][12][13][14][15] For example, the ESI-IM-MS analyses of the amb peptides shows that their formation of metal complexes are dependent on pH which is important for controlling the capture and release of tagged proteins in an IMAC column. A major attribute for ESI-IM-MS analyses is that it allows for the pH-dependent behavior of specific m/z conformers to be examined.…”

Electrically Switchable Nanolever Technology for the Screening of Metal-Chelating Peptides in Hydrolysates

“…Bioactive peptides with biological activities (e.g., antioxidant, antihypertensive, and anticancer activity) based on their amino acid composition and their primary sequence have been produced by enzymatic hydrolysis . Metal-ion chelation is a functional property commonly investigated in the literature. , Indeed, metals ions such as iron, zinc, calcium, and copper are essential for several biological functions such as enzyme catalysis, oxygen transport, and neurotransmitter release . However, their bioavailability is poor due to their low solubility in human fluids .…”

“…However, their bioavailability is poor due to their low solubility in human fluids . Metal-chelating peptides (MCPs) contribute to mineral absorption and bioavailability, thus regulating ion concentrations through selective bindings . Finally, iron- and copper-chelating peptideswhile complexing these transient metal ionscan act as indirect antioxidants by inhibiting in vivo the Fenton and Haber–Weiss reactions responsible for the formation of reactive oxygen species (including the hydroxyl radical) and subsequent radical chain reactions.…”

Collisional Dynamics Simulations Revealing Fragmentation Properties of Zn(II)-Bound Poly-Peptide

“…3 Ion mobility -mass spectrometry (IM-MS) [3][4][5] with molecular modelling and novel ion activation methods can be used to determine the accurate structure of these zinc containing metallopeptides. Previous studies of methanobactin 6,7 from Methylosinus trichosporium and analogue methanobactin (amb) peptides [3][4][5][8][9][10][11] show the IM-MS analyses in negative ion mode is particularly informative because it shows a distinct pH dependence for the metal binding and provides the protonation state of the acidic and basic binding sites, the charge of the metal ion, and the collision cross section of the conformer. The primary structures of the amb peptides include Pro as a hinge to position the His and Cys substituent groups for coordinating the metal ion ( Figure 1).…”