Figure 6
From: Expression and processing of mature human frataxin after gene therapy in mice
Detection and quantification of mature hFXN and its N-terminal tryptic peptides in the mouse liver following hFXN gene therapy. (a) Representative chromatograms from 2D-nano-UHPLC-PRM/HRMS analysis of mature hFXN N-terminal tryptic peptides in the liver of a mouse administered a high dose (1.8e13 gc/kg) of AAVrh.10hFXN. The red arrow indicates the retention time of the peptide. y-ion indicates the number of amino acids from the carboxy terminus that are present in this ion. Peptide retention times and relative amounts are shown on the left of the relevant signal. (b) Absolute concentrations of Mt and Extra-mt mature hFXN in mouse liver after administration of one of three different doses of AAVrh.10hFXN (low-dose = 1.8e12 gc/kg, n = 6; mid-dose = 5.7e12 gc/kg, n = 6; high-dose = 1.8e13 gc/kg, n = 8) or vehicle (n = 5). (c) Dose-dependent increase in total mature hFXN levels in mouse liver after administration of the same doses of AAVrh.10hFXN or vehicle (sample sizes as above). The dotted line represents endogenous total mature mFXN levels (14.9 ng/mg). (d) Relative amounts of mature hFXN proteoforms in mouse liver after administration of the same doses of AAVrh.10hFXN or vehicle (sample sizes as above). (b–d) Data are expressed as mean ± s.e.m. 2D-nano-UHPLC-PRM/HRMS = two-dimensional nano-ultra-high performance liquid chromatography-parallel reaction monitoring high-resolution mass spectrometry; AAVrh.10hFXN = adeno-associated virus rhesus serotype 10 encoding human frataxin; Extra-mt = extra-mitochondrial; hFXN = human frataxin; mFXN = mouse frataxin; Mt = mitochondrial; m/z = mass-to-charge ratio; NL = normalized signal level; s.e.m. = standard error of the mean.
