Review: Advances in the Accuracy and Traceability of Metalloprotein Measurements Using Isotope Dilution Inductively Coupled Plasma Mass Spectrometry

Figures & data

Figure 1. Work-flow diagram for species-unspecific isotope dilution mass spectrometry (SU-IDMS, left) compared with species-specific isotope dilution mass spectrometry (SS-IDMS, right) using a single separation column with UV-visible monitoring and online ICP-MS elemental quantitation. In species-unspecific IDMS, the isotopically enriched spike is continuously added post-separation, whereas in species-specific IDMS, the sample is mixed with an isotopically enriched species-specific spike before chromatographic separation.

Table 1. Isotopic spikes for species-specific IDMS used for the quantification of iron-containing metalloproteins – tabulated for easier comparison of spike manufacturing techniques.

Protein tested	Isotopic spike for species-specific ID	Stability studied	Reference
Transferrin	Standard of human Tf was incubated with a solution of ^57Fe in sodium bicarbonate for 30 min. The saturation was controlled with HPLC-UV. ^57Fe was characterized by reverse suIDMS.	24 h	^[^51^]
Transferrin	Apo-Tf was incubated with ^57Fe-citrate pH 7.4 (Tris, NH4HCO3, NaCl buffer) for 1 h, purified by ultrafiltration (cutoff 10 kDa, at 14,000g)	1 month	^[^52^]
Transferrin (iron saturation)	Based on Ref.^[^51^]: apo-Tf was mixed with NaHCO3; ^57FeCl3 solutions was then added in a concentration, which was adapted to the desired Tf saturation. The mixture was incubated for 60 min at room temperature.	24 h	^[^53^,^54^]
Transferrin (Tf-bound Fe)	Apo-Tf was incubated with ^54Fe spike in Tris carbonate buffer (pH 8.0) and purified using a PD-10 desalting column.	Not reported	^[^55^]
Non-Tf bound Fe as NTA complex	NTA-^57Fe was spiked into serum incubated with NTA.	Not reported	^[^59^]
Plant ferritin bound iron	^57Fe-labeled spike was generated by cloning and over expressing the Phaseolus vulgaris Fer gene in Escherichia coli and cultivating bacteria in a growth medium containing ^57FeCl2. The purification was undertaken as designed previously^[^61^]; gel filtration by a Sephadex® G-25 column; iron-containing fractions were eluted to the NaCl buffer (ca. 0.15 M), were pooled and concentrated 10–20-fold by ultrafiltration. Fer was further purified by size exclusion chromatography (Sephacryl® S-300 column), concentrated by ultrafiltration and stored at −20 °C.	≥5 months when kept in glycerol at −20 °C	^[^60^,^61^]
Human plasma Ferritin	^57Fe-Fer produced by biosynthesis, according to Ref.^[^61^] and in vitro labeling with the ^57Fe spike in the form of ^57Fe-citrate after cell lysis and heat treatment. Excess ^57FeCl2 was removed by ultrafiltration and purified by DEAE sepharose column followed by size exclusion chromatography using a Sephacryl® S-300 column. SDS-PAGE and native PAGE were used to assess spike purity.	Not reported	^[^62^]
Human serum ferritin and iron/ferritin ratio	Fer was isolated in immunoreaction with the formation of the bioconjugated complex (biotinylated antibody + Fer + antibody labeled with ruthenium) and collected on magnetic microparticles. Latex microparticles were used for Fe/Fer ratios.	Not reported	^[^58^]
Ferritin from murine blood serum and brain extracts	Commercial apo-Fer was enriched with ^57FeCl2: protein standard was diluted with 50 mM ammonium acetate buffer (pH 6.8) and mixed with ^57FeCl2 solution, vortexed for 1 min and incubated for 1 h at room temperature. Ultrafiltration (100 kDa cutoff) was used for the purification. IDMS was used to characterize the spike.	6 months	^[^66^]
Human hemoglobin in hemolyzed red blood cells; reference materials IRMM/IFCC-467 & JCCRM 912-2	^57Fe-Protohem and apo-HbA0 were prepared separately and reconstituted to ^57Fe-HbA0. Protoporphyrin IX was mixed with ^57Fe in acetate buffer solution and refluxed for 2 h, washed and lyophilized; The mixture of monomeric Hb α- and β-chains were dialyzed overnight against water, then against NaHCO3 buffer (24 h), then against phosphate buffer (6 h, pH 7.0). Finally, HbA was reconstituted under 3 h stirring on an ice bath. The spike was assessed by Raman spectroscopy and HPLC-ESI-MS.	42 h continuously; 5 months (when stored at −20ºC)	^[^74^]

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Data availability statement

There are no data association with this publication.