1. ASPP2 hydroxylation is detectable in vivo and the amount of hydroxylation depends on FIH-1 abundance.
ASPP2 hydroxylation is detectable in vivo and the amount of hydroxylation depends on FIH-1 abundance. The ASPP2 peptide GVNVNAADSDGWTPL resulting from chymotrypsin cleavage was detected by LC-MS/MS analysis both as a hydroxylated and a non-modified variant in ASPP2 purified from wild-type HEK293T cells, FIH-1-overexpressing cells and cells treated with the hydroxylase inhibitor DMOG. (A) MS spectra from an LC-MS/MS analysis of ASPP2 purified from samples with different amounts of FIH-1. The spectra are shown for the respective peak in the extracted ion chromatograms of the peptide variants (unmodified: [M+2H]2+ = 758.35; hydroxylated: [M+2H]2+ = 766.35). (B) Comparison of the relative amounts of unmodified and hydroxylated peptide. Peak areas from extracted ion chromatograms for the respective masses are shown. (C) MS/MS spectra confirming peptide identity and showing N986 as the site of modification.
Kirsten Janke et al. J Cell Sci 2013;126:
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