The analysis of whole blood by liquid chromatography-mass spectrometry using pig blood as a surrogate matrix may provide sensitive and specific information about the concentration of frataxin, a biomarker of Friedreich ataxia (FA), according to a research study published in Analytical Chemistry.

To analyze frataxin-M and frataxin-E in whole blood samples, a surrogate matrix was required for the preparation of standard curves, quality control samples, inter- and intraday samples, and freeze−thaw samples during the validation process.

For the prevention of unfavorable endogenous interference, it was necessary to use whole blood from a mammal that expresses different frataxin proteoforms from human frataxin. The study authors analyzed sequences from 14 mammalian species and found pig blood to be a viable surrogate matrix option.

The pig-derived frataxin forms could be differentiated from the human proteoforms by ultrahigh-performance liquid chromatography−multiple reaction monitoring/mass spectrometry (UHPLC-MRM/MS) analysis. Moreover, pig frataxin would not be a substrate for the immobilized monoclonal antibody used for immunoprecipitation of the whole blood and could be eliminated during the immunoprecipitation step used to isolate human frataxin.

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After successfully validating stable isotope dilution UHPLC-MRM/MS assays for frataxin-M and frataxin-E using pig blood as the surrogate matrix, the researchers analyzed blood samples from 25 healthy controls, 25 heterozygous carriers of the FXN gene with guanine-adenine-adenine (GAA) repeats, and 25 patients with FA.

According to the results, there was no overlap in frataxin proteoform levels between healthy controls and patients with FA. The method presented 100% specificity and 100% sensitivity for distinguishing healthy controls from patients with FA.

Moreover, the analysis showed linear correlations between frataxin levels and the GAA repeat length and age of onset, although the correlation of frataxin-E remained more significant than the correlation of frataxin-M.

“These findings auger well for using the assay to monitor therapeutic interventions that are targeted to upregulate frataxin levels as well as for monitoring the natural history of the disease,” Rojsajjakul and colleagues wrote. “Furthermore, the ability to use a triple-quadrupole mass spectrometer rather than a high-resolution instrument will greatly facilitate these studies.”

As frataxin-M is expressed in blood cells that contain mitochondria and frataxin-E is only found in erythrocytes, the analysis of whole blood provides information on the concentration of both proteoforms without having to isolate the individual cell types.


Rojsajjakul T, Wu L, Grady CB, et al. Liquid chromatography–mass spectrometry analysis of frataxin proteoforms in whole blood as biomarkers of the genetic disease Friedreich’s ataxiaAnal Chem. Published online February 17, 2023. doi:10.1021/acs.analchem.3c00091