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 multiplex assay by quantifying the FANCD2 proteoforms following mitomycin-c treatment in an isogenic pair of FancA-corrected and uncorrected cell lines, as well as primary peripheral blood mononuclear cells from Fanconi Anemia patients. Additionally, we demonstrate detection of endogenous FANCD2 monoubiquitination in human breast cancer tissue. The immuno-MRM assay provides a potential functional diagnostic for patients with Fanconi Anemia with defects in the upstream FA complex or FANCD2, and a potential test for predicting sensitivity to DNA cross-linking agents in human cancers.

Established protocols were used to develop an immuno-MRM assay to the tryptic peptide containing the site of monoubiquitination on FANCD2. Anti-peptide monoclonal antibodies were generated using the modified peptide (containing a –GG remnant on lysine 561) as the immunogen sequence. Synthetic peptides (modified and unmodified) were used to optimize MRM mass spectrometric parameters. Characterization experiments were performed in a pool of lymphoblast cell lines +/- exposure to ionizing radiation to induce DNA damage. Proof-of-principle experiments were conducted in an isogenic uncorrected/corrected pair of lymphoblast cell lines prepared from the FANCA-deficient HSC72 cell line, peripheral blood mononuclear cells isolated from patients with Fanconi Anemia, and fresh-frozen breast tumor tissue. Assay workflow includes protein extraction, trypsin digestion, peptide immunoaffinity enrichment, and MRM.