Electrospray-Induced Mass Spectrometry Is Not Suitable for Determination of Peptidic Cu(II) Complexes

Abstract

The toolset of mass spectrometry (MS) is still expanding, and the number of metal ion complexes researched this way is growing. The Cu(II) ion forms particularly strong peptide complexes of biological interest which are frequent objects of MS studies, but quantitative aspects of some reported results are at odds with those of experiments performed in solution. Cu(II) complexes are usually characterized by fast ligand exchange rates, despite their high affinity, and we speculated that such kinetic lability could be responsible for the observed discrepancies. In order to resolve this issue, we selected peptides belonging to the ATCUN family characterized with high and thoroughly determined Cu(II) binding constants and re-estimated them using two ESI-MS techniques: standard conditions in combination with serial dilution experiments and very mild conditions for competition experiments. The sample acidification, which accompanies the electrospray formation, was simulated with the pH−jump stopped-flow technique. Our results indicate that ESI-MS should not be used for quantitative studies of Cu(II)−peptide complexes because the electrospray formation process compromises the entropic contribution to the complex stability, yielding underestimations of complex stability constants.

Repurposing a peptide antibiotic as a catalyst: a multicopper–daptomycin complex as a cooperative O–O bond formation and activation catalyst

“…Detailed conditions for the preparation of Cu n -dap complex samples and ESI-HRMS analysis are provided in the ESI (section 7 †). Although the decrease in pH during ionization resulted in the demetallation of the Cu 4 -dap complex in the mass chamber, 44 the HRMS peaks at m/z 1880.37…”

“…Detailed conditions for the preparation of Cu n –dap complex samples and ESI-HRMS analysis are provided in the ESI (section 7†). Although the decrease in pH during ionization resulted in the demetallation of the Cu 4 –dap complex in the mass chamber, 44 the HRMS peaks at m / z 1880.37 ([dap L + 4Cu − 9H] −1 , z = −1) and 939.67 ([dap L + 4Cu − 10H] −2 , z = −2) support the hypothesis that the tetranuclear complex was formed in solution (Fig. S7–S9†).…”

An Overlooked Hepcidin-cadmium Connection

“…The samples were injected on Q-exactive UHMR mass spectrometer designed to preserve non-covalent bonds. One should always be wary of potential errors and differences in ionization, bonds not being carried over to gas phase or general lack of solvent during measurement [23]. However, in this case Cd(II) is not a fast exchanging metal ion and the peptides were similar enough to consider this technique.…”

Interactions of neurokinin B with copper(ii) ions and their potential biological consequences

“…28 The latter result cannot be treated as definitive, however, because mass spectrometry of Cu(II)-peptide complexes is prone to a number of experimental issues which compromise the reliability of results. 29 In this study, we re-investigated the Cu(II) complexation by NKB using potentiometry, spectroscopic methods, and DFT calculations. NKB is very poorly soluble in water and its biological actions are associated with membranes via the receptor binding and perhaps directly as well.…”

“…28 The latter result cannot be treated as definitive, however, because mass spectrometry of Cu( ii )–peptide complexes is prone to a number of experimental issues which compromise the reliability of results. 29…”