Targeted Mass Spectrometry Enables Quantification of Novel Pharmacodynamic Biomarkers of ATM Kinase Inhibition

Abstract

The ATM serine/threonine kinase (HGNC: ATM) is involved in initiation of repair of DNA double-stranded breaks, and ATM inhibitors are currently being tested as anti-cancer agents in clinical trials, where pharmacodynamic (PD) assays are crucial to help guide dose and scheduling and support mechanism of action studies. To identify and quantify PD biomarkers of ATM inhibition, we developed and analytically validated a 51-plex assay (DDR-2) quantifying protein expression and DNA damage-responsive phosphorylation. The median lower limit of quantification was 1.28 fmol, the linear range was over 3 orders of magnitude, the median inter-assay variability was 11% CV, and 86% of peptides were stable for storage prior to analysis. Use of the assay was demonstrated to quantify signaling following ionizing radiation-induced DNA damage in both immortalized lymphoblast cell lines and primary human peripheral blood mononuclear cells, identifying PD biomarkers for ATM inhibition to support preclinical and clinical studies.

Fanconi anemia-isogenic head and neck cancer cell line pairs - a basic and translational science resource

“…This approach allowed us to simultaneously detect and quantify FANCD2 expression and monoubiquitylation status, together with the abundance and post-translational modification status of peptides located in 65 additional DNA damage response proteins including 9 other FANC proteins. The outline of these experiments is shown in Figure 5A (26–28), in which we were able to quantify 98 of 129 potential target peptides using experimentally-defined lower limits for quantification (LOQ)(Figure 5B and 5C, Table S3).…”

“…In brief, triplicate 10 cm dishes were seeded with 2 - 5 × 10 6 cells prior to the addition of 0.2 µM mitomycin C for 24 hrs. Whole cell lysates were prepared and processed in a blinded fashion as previously described, using two antibody panels that targeted 126 peptides and 59 post-translational modification sites (26–28). MRM-MS data were analyzed using Skyline (29), with manual review of peak integrations to confirm and align peptide transitions of endogenous and stable mass isotope-labeled peptide standards.…”

“…Peptide immunoenrichment coupled with targeted multiple reaction monitoring mass spectrometry (immuno-MRM) was used as a complementary approach to detect and quantify the expression and post-translational modification status of FANCD2 and additional DNA damage response and repair (DDR) proteins prior to and after MMC treatment. The two immuno-MRM panels used for these analyses target 126 peptides and 62 post-translational modification sites in 66 DDR proteins including 10 FANC proteins (36–38).…”

“…# 28-9513-79), and the flow-through from enrichment of the first antibody panel was used as input for the second antibody panel purification. All eluates were frozen and analyzed independently by mass spectrometry as previously described (36–38), using independent runs for the enriched peptides from the two panels.…”

“…The LC gradient was delivered at 300 nL/min, and consisted of a linear gradient of mobile phase B (90% acetonitrile and 0.1% formic acid in water) from 3-14% B over 1 min, 14-34% B over 20 min and 34-90% B over 2 min followed by re-equilibration at 3% B on a 15 cm × 75 m chip column (Reprosil AQ C18 particles, 3 m; Dr. Maisch, Germany). Parameters for collision energy (CE) were taken from prior optimization of synthetic peptides (36,38). Scheduled MRM transitions used a retention time window of 150 sec and a cycle time of 1.0 sec to enable sufficient point sampling across peaks to allow MRM data to be quantified using Skyline (39)(see: https://skyline.ms/project/home/software/skyline/begin.view).…”