PUBLICATION
Stable Isotope Labeling in Bacteria Enables Characterization and Quantification of Frataxin Protein in a Friedreich's Ataxia Zebrafish Model
- Authors
- Rojsajjakul, T., Do, W., Wilson, R.B., Blair, I.A.
- ID
- ZDB-PUB-250625-14
- Date
- 2025
- Source
- Analytical chemistry 97: 13779-13788 (Journal)
- Registered Authors
- Keywords
- none
- MeSH Terms
-
- Animals
- Chromatography, High Pressure Liquid
- Disease Models, Animal
- Escherichia coli*/genetics
- Escherichia coli*/metabolism
- Frataxin
- Friedreich Ataxia*/genetics
- Friedreich Ataxia*/metabolism
- Humans
- Iron-Binding Proteins*/analysis
- Iron-Binding Proteins*/genetics
- Iron-Binding Proteins*/metabolism
- Isotope Labeling*
- Mass Spectrometry
- Zebrafish
- PubMed
- 40554456 Full text @ Anal. Chem.
Citation
Rojsajjakul, T., Do, W., Wilson, R.B., Blair, I.A. (2025) Stable Isotope Labeling in Bacteria Enables Characterization and Quantification of Frataxin Protein in a Friedreich's Ataxia Zebrafish Model. Analytical chemistry. 97:13779-13788.
Abstract
Friedreich's ataxia (FRDA) is a neurodegenerative and cardiodegenerative genetic disorder caused primarily by homozygous mutations in the FXN gene, resulting in decreased expression of human mature frataxin (hFXN-M) protein. To test potential new drugs, we developed mutant zebrafish with a deficiency in zebrafish FXN-M (zFXN-M) production by introducing targeted mutations in the z-fxn gene. To validate this model, it was necessary to characterize and quantify zFXN-M protein, but zFXN-M protein could not be detected by Western blot in zebrafish lysates. We developed an alternative strategy involving the use of a stable isotope-labeled internal standard coupled with analysis by high-sensitivity ultrahigh-performance liquid chromatography-multiple reaction monitoring-mass spectrometry (UHPLC-MRM/MS). The endogenous zFXN-M in an internal standard prepared using stable isotope labeling by amino acids in cell culture (SILAC) would have obscured low levels of zFXN-M. In contrast, stable isotope labeling in bacteria (SILIB) provided fully labeled [13C,15N]-zFXN-M with almost undetectable amounts of endogenous protein contamination. This facilitated characterization of amol levels of zFXN-M in zebrafish embryos (120.9 ± 20.1 amol/embryo) and its quantification in intact wild-type fish with levels of 2.26 ± 0.44 ng/mg protein or 145.2 ± 24.5 pg/mg tissue. Recovery of zFXN-M was <10% when the SILIB internal standard was added after isolation, when compared with before isolation. UHPLC-MRM/MS with a SILIB internal standard was the only way to validate zebrafish heterozygous for a knockout mutation in zFXN as a model for FRDA, illustrating its utility for the characterization and quantification of very low abundance tissue proteins.
Genes / Markers
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Orthology
Engineered Foreign Genes
Mapping