Multi-isotope-dilution quantification in metabolome- and proteome-wide speciation analysis of metal complexes – MIDQuant

The objective of the project is to develop a new concept for the quantification in trace metal speciation analysis, and, in particular, in large-scale analysis of metal-complexes with low molecular mass ligands (metallo-metabolomics) and proteins (metalloproteomics).

The concept is based on the addition of a mixture of isotopically enriched metal species (in the case of covalent complexes) or metal ions (multi-isotopic metal spike) in the case of coordination complexes. It is followed by the measurement, by high-resolution high accurate electrospray mass spectrometry (Orbitrap MS and Fourier Transform Ion Cyclotron Resonance MS), of the changes in the metal isotopic composition, simultaneously or quasi-simultaneously (after HPLC), in each and every metal-complex present.

The first practical consequence of the enriched isotope addition matching the original (unknown) speciation or isotopic exchange will be the possibility of mining large MS data sets for the modified isotopic patterns allowing the identification of the subsets of metal-species present and, subsequently, characterize them using the accurate molecular mass and MSn fragmentation data.

On the level of metrology, the project will take advantage of the quasi-non-vulnerability of the isotope ratio measurements to the varying chromatographic recovery, stability, and matrix effects and exploit it for accurate relative or absolute large-scale quantification in speciation analysis. We will explore the potential of Orbitrap and FTICR mass spectrometry for the isotope ratio measurement in the high-resolution mode (>500,000) simultaneously for hundreds of species with precision values potentially approaching the values routinely achieved with magnetic sector instruments. The concept will be first applied to the accurate quantification of tens of covalent species (selenium metabolome in Se-enriched yeast) in large scale speciation analysis against a well-defined standard reference material.

The project will investigate to which degree metal-complexes with biological ligands are prone to exchange isotopic ions. On the basis of preliminary experiments, we expect that spike added in excess, will consume first the excess of ligand, and then exchange with the naturally present metal ions in the complexes. The measurement of the changes in the isotope ratios (which are independent of the electrospray sensitivity) combined with the knowledge of the amounts of elements initially present and isotopic spike added should allow the quantification of the individual metal-species considerably reducing the need for authentic standards of each of the metal-ligand complex present.

The different rate of isotopic exchange will be investigated as a tool to distinguish true metalloproteins (metal as coordination center, potentially thermodynamically and kinetically stable complexes) and metal-binding proteins (potentially labile complexes) on the level of the proteome.