Ultratrace Metal Speciation Analysis by Coupling of Sector-Field ICP-MS to High-Resolution Size Exclusion and Reversed-Phase Liquid Chromatography

Abstract

Techniques for metal speciation analysis with subnanomolar (ppt) detection limits in complex matrices, with simultaneous quantification of matrix elements, have become a necessity for investigating targets of trace metal binding to macromolecules and pigments at environmentally relevant concentrations. In this work we optimized the analysis of such metal binding in a custom-built HPLC-ICP-MS system. Key elements of the optimization were the choice of components for the metal-free HPLC-DAD system and sector-field ICP-MS detection (ICF-sfMS) with desolvating injection and optimization of sample handling. Protein analysis was done using ammonium bicarbonate buffer and size exclusion chromatography (SEC-ICP-sfMS), with possible addition of anion exchange chromatography. Detection of metal exchange in pigments (chlorophylls and bacteriochlorophylls) was based on reversed-phase chromatography with a methanol-acetone gradient and coupling to the ICP-sfMS via a dedicated organic matrix interface (RPC-ICP-sfMS). The resulting HPLC-DAD-ICP-sfMS system has detection limits in the picomolar range in protein buffer, limited by the maximal achievable purity of buffers/solvents and not by system sensitivity. Tests for method optimization showed that sonication, meant to increase protein solubilization, leads to artifacts of metal loss from metalloproteins. Examples for Cd binding to soybean proteins and chlorophyll, Cr binding to Arabidopsis thaliana proteins, La binding to Desmodesmus quadricauda proteins, and Cu binding to Rhodospirillum rubrum proteins and pigments are shown. These application examples demonstrate that the system is sensitive enough to detect binding of metals to proteins and pigments at background concentration levels of typical nutrient solutions made from analytical grade chemicals, equivalent to ultratrace metal concentrations in nonpolluted environments.

Nodule-specific Cu+-chaperone NCC1 is required for symbiotic nitrogen fixation inMedicago truncatularoot nodules

“…Metal-EDTA complexes in same buffer as the protein extracts were used to calibrate concentration determination. A gel filtration calibration standard (Bio-rad with added PABA) was used to determine size and molecular weight, calibration of metal concentrations in the ICP-MS chromatograms was performed as described in Küpper et al (2019). Proteins were identified in the Proteomic Unit of Universidad Complutense de Madrid (Spain).…”

Micronutrient homeostasis in plants for more sustainable agriculture and healthier human nutrition

“…Finally, metal-sensitive fluorescent probes, such as Förster resonance energy transfer (FRET) sensors, dynamically monitor labile micronutrient pools in vivo ( Lanquar et al , 2014 ). In addition, analytical chemistry methods combining chromatography with mass spectrometry and elemental analysis, such as size-exclusion chromatography (SEC)-ICP-MS, have identified metal complexes with small molecules or with proteins ( Cakmak et al , 2010a ; Flis et al , 2016 ; Küpper et al , 2019 ). Ultimately, the development of these approaches should allow the determination of the complete spectrum of micronutrient metal complexes with small molecules (metallome), and the full complement of metalloproteins (metalloproteome) for any given biological sample.…”

Atomic spectrometry update: review of advances in elemental speciation

“…Two unrelated studies on the speciation of trace metals and Pd-nanoparticles in plants have highlighted improvements in chromatographic resolution, elemental sensitivity and the use of orthogonal detection methods , which make it possible to investigate trace metal binding to proteins and pigments at environmentally relevant concentrations and the investigation of ligands generated in response to Pd exposure. Küpper et al 185 have designed a sophisticated HPLC-SF-ICP-MS system with a metal-free flow path and a switching system so that high concentration calibration standards do not come into contact with the chromatographic columns used, which can lead to number of problems due to metals being absorbed onto the column packing material. In unison, they also purified the buffers, using Chelex-100, to reduce the metal content and in this way were able to achieve very low baseline signals.…”