Targeted mass spectrometry enables robust quantification of FANCD2 mono-ubiquitination in response to DNA damage.

ABSTRACT: The Fanconi anemia pathway is an important coordinator of DNA repair pathways and is particularly relevant to repair of DNA inter-strand crosslinks. Central to the pathway is monoubiquitination of FANCD2, requiring the function of multiple proteins in an upstream Fanconi core complex. We present development and analytical characterization of a novel assay for quantification of unmodified and monoubiquitinated FANCD2 proteoforms, based on peptide immunoaffinity enrichment and targeted multiple reaction monitoring mass spectrometry (immuno-MRM). The immuno-MRM assay is analytically characterized using fit-for-purpose method validation. The assay linear range is >3 orders of magnitude with total repeatability <16% CV. In proof-of-principle experiments, we demonstrate application of the multip

SUBMITTER: Whiteaker JR 

PROVIDER: S-EPMC5911425 | biostudies-literature | 2018 May

REPOSITORIES: biostudies-literature