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. 2023 Dec 1:1231:123921.
doi: 10.1016/j.jchromb.2023.123921. Epub 2023 Nov 10.

Improved methods for the detection of heme and protoporphyrin IX adducts and quantification of heme B from cytochrome P450 containing systems

Robert D Pelletier  1 , Allan E Rettie  2 , John P Kowalski  2
Affiliations

Improved methods for the detection of heme and protoporphyrin IX adducts and quantification of heme B from cytochrome P450 containing systems

Robert D Pelletier et al. J Chromatogr B Analyt Technol Biomed Life Sci. .

Abstract

Heme B is a critical prosthetic group for the function of numerous proteins including the cytochrome P450 (CYP) family of enzymes. CYP enzymes are involved in the metabolism of endogenous and xenobiotic molecules that are of central interest in drug development. Formation of reactive metabolites by CYPs can lead to heme modification and destruction of the enzyme. The structure of the adducted heme can provide key information on the mechanism of inactivation, which is of great interest during preclinical drug discovery. Historically, techniques to extract the modified heme or protoporphyrin IX species involved harsh extraction conditions and esterification of propionate groups to aid chromatography. We have developed a simplified extraction method and LC/MS chromatography system that does not require derivatization to quantify heme B and identify modified heme B species from multiple CYP-containing matrices. The method uses mass defect filter triggered data dependent MS2 scans to rapidly identify heme and protoporphyrin IX adducts. These methods may also be useful for the analysis of other heme variants and hemoproteins.

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Conflict of interest statement

Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Figures

Figure 1.
Figure 1.
Spectra and extracted ion current of heme B extracted from 250 nM CYP2E1 with 500 nM D4-Heme B. (A) Centroided spectra from 2.27 minute peak. (B) Extracted ion current of TOF MS for heme B and the major isotope for the D4-heme B standard. (C) PIS of heme B (616.18 m/z). (D) PIS of D4-heme B (621.21 m/z).
Figure 2.
Figure 2.
Heme and protoporphyrin IX related adducts from incubations of ABT with HLM (A) Extracted ion current of ABT heme and protopophyrin adducts. (B) PIS of 3.17 min peak of 637.28 m/z. (C) PIS of 3.29 min peak of 637.28 m/z. (D) PIS of 3.58 min peak of 692.21 m/z. (E) Proposed fragmentation of N,N-bridged protoporphyrin IX bridged adducts. (F) Proposed fragmentation of benzyl heme B adducts.
Figure 3.
Figure 3.
Heme and protoporphyrin IX related adducts from incubations of DDC with HLM (A) Extracted ion current of DDC protoporphyrin adducts. (B) PIS of 3.31 min peak of 577.28 m/z. (C) Proposed fragmentation of DDC protoporphyrin adducts.
Figure 4.
Figure 4.
Heme and protoporphyrin IX related adducts from incubations of ECH with HLM (A) Extracted ion current of 1-ethynylcyclohexanol protopophyrin adducts. (B) PIS of 2.90 min peak of 703.35 m/z. (C) PIS of 3.10 min peak of 703.35 m/z. (D) Proposed fragmentation of ECH protoporphyrin adducts.
Figure 5.
Figure 5.
Heme and protoporphyrin IX related adducts from incubations of 1-octene with HLM (A) Extracted ion current of 1-octene protoporphyrin adducts. (B) PIS of 3.56 min peak of 691.38 m/z. (C) Proposed fragmentation of 1-octene protoporphyrin adducts.
Figure 6.
Figure 6.
Heme and protoporphyrin IX related adducts from incubations of phenylacetylene with HLM (A) Extracted ion current of phenylacetylene heme B and protoporphyrin adducts. (B) PIS of 3.81 min peak of 681.31 m/z. (C) Proposed fragmentation of phenylacetylene protoporphyrin adducts.
Figure 7.
Figure 7.
Heme and protoporphyrin IX related adducts from incubations of phenylhydrazine with rabbit liver microsomes (A) Extracted ion current of phenylhydrazine heme B and verdoheme adducts. (B) PIS of 3.39 min peak of 693.22 m/z. (C) PIS of 3.44 min peak of 693.22 m/z. (D) Proposed fragmentation of phenyhydrazine heme B adducts. (E) Proposed fragmentation of phenyhydrazine verdoheme B adducts.
Figure 8.
Figure 8.
Extracted ion current of ABT and A8A heme and protopophyrin adducts from incubations with rCYP4Z1. Only species with a m/z of 637.28 were detected in the ABT incubation while no other heme or PPIX were detected in any of the other incubations.
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