Stable Isotope Labeling in Bacteria Enables Characterization and Quantification of Frataxin Protein in a Friedreich's...

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.