Iron Redox Speciation Analysis Using Capillary Electrophoresis Coupled to Inductively Coupled Plasma Mass Spectrometry (CE-ICP-MS)

Abstract

Neuronal iron dyshomeostasis occurs in multiple neurodegenerative diseases. Changes in the Fe(II)/Fe(III) ratio toward Fe(II) is closely related to oxidative stress, lipid peroxidation, and represents a hallmark feature of ferroptosis. In particular for body fluids, like cerebrospinal fluid (CSF), reliable quantitative methods for Fe(II)/(III) redox-speciation analysis are needed to better assess the risk of Fe(II)-mediated damage in brain tissue. Currently in the field of metallomics, the most direct method to analyze both iron species is via LC-ICP-MS. However, this Fe(II)/(III) speciation analysis method suffers from several limitations. Here, we describe a unique method using capillary electrophoresis (CE)-ICP-MS for quantitative Fe(II)/(III) speciation analysis that can be applied for cell lysates and biofluid samples. Compared to LC, CE offers various advantages: (1) Capillaries have no stationary phase and do not depend on batch identity of stationary phases; (2) Replacement of aged or blocked capillaries is quick with no performance change; (3) Purge steps are effective and short; (4) Short sample analysis time. The final method employed 20 mM HCl as background electrolyte and a separation voltage of +25 kV. In contrary to the LC-method, no complexation of Fe-species with pyridine dicarboxylic acid (PDCA) was applied, since it hampered separation. Peak shapes and concentration detection limits were improved by combined conductivity-pH-stacking achieving 3 μg/L detection limit (3σ) at 13 nL injection volume. Calibrations from LOD—150 μg/L were linear [r2[Fe(II)] = 0.9999, r2[Fe(III)] = 0.9951]. At higher concentrations Fe(II) curve flattened significantly. Measurement precision was 3.5% [Fe(II) at 62 μg/L] or 2.2% [Fe(III) at 112 μg/L] and migration time precision was 2% for Fe(III) and 3% for Fe(II), each determined in 1:2 diluted lysates of human neuroblastoma cells. Concentration determination accuracy was checked by parallel measurements of SH-SY5Y cell lysates with validated LC-ICP-MS method and by recovery experiments after standard addition. Accuracy (n = 6) was 97.6 ± 3.7% Fe(III) and 105 ± 6.6%Fe(II). Recovery [(a) +33 μg/L or (b) +500 μg/L, addition per species] was (a): 97.2 ± 13% [Fe(II)], 108 ± 15% [Fe(III)], 102.5 ± 7% (sum of species), and (b) 99±4% [Fe(II)], 101 ± 6% [Fe(III)], 100 ± 5% (sum of species). Migration time shifts in CSF samples were due to high salinity, but both Fe-species were identified by standard addition.

BNIP3 promotes HIF‐1α‐driven melanoma growth by curbing intracellular iron homeostasis

“…In line with this, only BNIP3‐depleted cells—but not autophagy‐compromised cells—displayed a significantly higher content of intracellular Fe 2+ as imaged and quantitated by FerroOrange, a probe reacting specifically with intracellular Fe 2+ ions (Fig 6A). Furthermore, to validate this finding we performed highly sensitive capillary electrophoresis combined with inductively coupled plasma mass spectrometry(CE‐ICP‐MS), from shCntl, shBNIP3, and shATG5 melanoma cells, which allowed for quantitative Fe 2+ /Fe 3+ speciation analysis (Michalke et al, 2019). This analysis showed that while the total iron content across these cell lines remained constant (Fig 6B; Appendix Table S3), shBNIP3 cells harbored a significantly higher proportion of Fe 2+ when compared to Fe 3+ (Fig 6B, Appendix Table S3).…”

“…Cell lysates were centrifuged at 10,000 g for 10 min, and supernatants were sent on dry ice to Helmholtz Center Munich. Speciation and quantification of Fe 2+ , Fe 3+ , and total iron were performed by capillary electrophoresis inductively coupled plasma mass spectrometry (CE‐ICP‐MS) as described previously (Michalke et al, 2019). Briefly, samples were analyzed on a "PrinCe 706” CE system equipped with an uncoated capillary (85 cm × 50 µm ID) and a laboratory‐constructed CE‐ICP‐MS interface for element selective iron quantification of separated iron redox species at ICP‐DRC‐MS.…”

Dystrophic microglia are a disease associated microglia morphology in the human brain

“…More work is needed to directly measure iron burden in FTL positive microglia from brains with and without neurodegenerative pathology. For instance, Fe(II) and Fe(III) could be measured on FACS sorted microglia from the postmortem tissue using capillary electrophoresis coupled-to-inductively-coupled plasma mass spectrometry (CE-ICP-MS)[35].…”

A crosstalk between hepcidin and IRE/IRP pathways controls ferroportin expression and determines serum iron levels in mice

“…Quantification of liver iron content (LIC). Total liver iron was quantified by using the ferrozine assay (11) or inductively coupled plasma mass spectrometry (ICP-MS) (38).…”