In seawater, copper (Cu) is almost entirely complexed by organic ligands, many of which are resolvable by liquid chromatography but remain structurally unelucidated.  The microbial sources of these ligands are unknown, as are the roles they play in the cycling and bioavailability of Cu.  In order to better characterize the biological sources of Cu ligands, we grew laboratory cultures of the model marine diatom <i>Phaeodactylum tricornutum</i>CCMP632 and analyzed the culture medium.  Using liquid chromatography (LC) coupled to an inductively coupled plasma mass spectrometer (ICP-MS) we found this diatom produces a suite of Cu ligands, well-resolved by LC, with a range of polarities similar to what has been reported for marine surface waters.  Parallel analyses using LC coupled to high-resolution electrospray ionization mass spectrometry (LC-ESIMS) yields a suite of co-eluting ions at the retention times of the Cu ligands.  We must then determine which ion contains Cu from this list.  To find the mass of the Cu ligands we used an algorithm that identifies isotopologues with the correct mass difference and relative abundance of naturally occurring Cu-63 and Cu-65.  This approach yielded a suite of potential molecular ions, but isobaric interferences to the fine structure of the Cu isotopic distribution from other isotopes made positive assignments of the Cu containing molecular ion difficult. Therefore, we applied a second approach that uses high-energy collisional dissociation to fragment the Cu ligand such that the Cu ion is released from the ligand complex.  This product-precursor approach allows us to directly observe the Cu ion, clearly demonstrating that the precursor ion is a Cu containing compound.  Previous culture studies have shown that the production of Cu ligands is widespread among marine microorganisms, however molecular level characterization of these compounds has been lacking.  Our new approach seeks to address this gap in our knowledge, which is a step towards determining the source and identities of naturally occurring Cu ligands in seawater.

Mass spectral characterization of copper ligands produced by the marine diatom, <i>phaeodactylum tricornutum</i>

ACS Fall 2021

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Mass spectral characterization of copper ligands produced by the marine diatom, phaeodactylum tricornutum