Immobilized Metal Affinity Chromatography Coupled to Multiple Reaction Monitoring Enables Reproducible Quantification of Phospho-signaling.

A major goal in cell signaling research is the quantification of phosphorylation pharmacodynamics following perturbations. Traditional methods of studying cellular phospho-signaling measure one analyte at a time with poor standardization, rendering them inadequate for interrogating network biology and contributing to the irreproducibility of preclinical research. In this study, we test the feasibility of circumventing these issues by coupling immobilized metal affinity chromatography (IMAC)-based enrichment of phosphopeptides with targeted, multiple reaction monitoring (MRM) mass spectrometry to achieve precise, specific, standardized, multiplex quantification of phospho-signaling responses. A multiplex immobilized metal affinity chromatography- multiple reaction monitoring assay targeting phospho-analytes responsive to DNA damage was configured, analytically characterized, and deployed to generate phospho-pharmacodynamic curves from primary and immortalized human cells experiencing genotoxic stress. The multiplexed assays demonstrated linear ranges of ≥3 orders of magnitude, median lower limit of quantification of 0.64 fmol on column, median intra-assay variability of 9.3%, median inter-assay variability of 12.7%, and median total CV of 16.0%. The multiplex immobilized metal affinity chromatography- multiple reaction monitoring assay enabled robust quantification of 107 DNA damage-responsive phosphosites from human cells following DNA damage. The assays have been made publicly available as a resource to the community. The approach is generally applicable, enabling wide interrogation of signaling networks.

Peptide Immunoaffinity Enrichment and Targeted Mass Spectrometry Enables Multiplex, Quantitative Pharmacodynamic Studies of Phospho-Signaling.

In most cell signaling experiments, analytes are measured one Western blot lane at a time in a semiquantitative and often poorly specific manner, limiting our understanding of network biology and hindering the translation of novel therapeutics and diagnostics. We show the feasibility of using multiplex immuno-MRM for phospho-pharmacodynamic measurements, establishing the potential for rapid and precise quantification of cell signaling networks. A 69-plex immuno-MRM assay targeting the DNA damage response network was developed and characterized by response curves and determinations of intra- and inter-assay repeatability. The linear range was ≥ 3 orders of magnitude, the median limit of quantification was 2.0 fmol/mg, the median intra-assay variability was 10% CV, and the median interassay variability was 16% CV. The assay was applied in proof-of-concept studies to immortalized and primary human cells and surgically excised cancer tissues to quantify exposure-response relationships and the effects of a genomic variant (ATM kinase mutation) or pharmacologic (kinase) inhibitor. The study shows the utility of multiplex immuno-MRM for simultaneous quantification of phosphorylated and nonmodified peptides, showing feasibility for development of targeted assay panels to cell signaling networks.

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

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.

A Multiplexed Mass Spectrometry-Based Assay for Robust Quantification of Phosphosignaling in Response to DNA Damage.

A lack of analytically robust and multiplexed assays has hampered studies of the large, branched phosphosignaling network responsive to DNA damage. To address this need, we developed and fully analytically characterized a 62-plex assay quantifying protein expression and post-translational modification (phosphorylation and ubiquitination) after induction of DNA damage. The linear range was over 3 orders of magnitude, the median inter-assay variability was 10% CV and the vast majority (∼85%) of assays were stable after extended storage. The multiplexed assay was applied in proof-of-principle studies to quantify signaling after exposure to genotoxic stress (ionizing radiation and 4-nitroquinoline 1-oxide) in immortalized cell lines and primary human cells. The effects of genomic variants and pharmacologic kinase inhibition (ATM/ATR) were profiled using the assay. This study demonstrates the utility of a quantitative multiplexed assay for studying cellular signaling dynamics, and the potential application to studies on inter-individual variation in the radiation response.

The human salivary proteome is radiation responsive.

In the event of a nuclear incident in a heavily populated area, the surge in demand for medical evaluation will likely overwhelm our emergency care system, compromising our ability to care for victims with life-threatening injuries or exposures. Therefore, there exists a need for a rapidly deployable biological assay for radiation exposure that can be performed in the field by individuals with little to no medical training. Saliva is an attractive biofluid for this purpose, due to the relative ease of its collection and the wide array of biomolecules it contains. To determine whether the human salivary proteome is responsive to ionizing radiation exposure, we characterized the abundances of salivary proteins in humans before and after total body irradiation. Using an assay panel targeting 90 analytes (growth factors, chemokines and cytokines), we identified proteins that were significantly radiation responsive in human saliva. The responses of three proteins (monocyte chemo-attractant protein 1, interleukin 8 and intercellular adhesion molecule 1) were confirmed using independent immunoassay platforms and then verified and further characterized in 130 saliva samples from a completely independent set of 38 patients undergoing total body irradiation. The results demonstrate the potential for detecting radiation exposure based on analysis of human saliva.

Blood-based detection of radiation exposure in humans based on novel phospho-Smc1 ELISA.

The structural maintenance of chromosome 1 (Smc1) protein is a member of the highly conserved cohesin complex and is involved in sister chromatid cohesion. In response to ionizing radiation, Smc1 is phosphorylated at two sites, Ser-957 and Ser-966, and these phosphorylation events are dependent on the ATM protein kinase. In this study, we describe the generation of two novel ELISAs for quantifying phospho-Smc1(Ser-957) and phospho-Smc1(Ser-966). Using these novel assays, we quantify the kinetic and biodosimetric responses of human cells of hematological origin, including immortalized cells, as well as both quiescent and cycling primary human PBMC. Additionally, we demonstrate a robust in vivo response for phospho-Smc1(Ser-957) and phospho-Smc1(Ser-966) in lymphocytes of human patients after therapeutic exposure to ionizing radiation, including total-body irradiation, partial-body irradiation, and internal exposure to (131)I. These assays are useful for quantifying the DNA damage response in experimental systems and potentially for the identification of individuals exposed to radiation after a radiological incident.

North Queensland midwives' experience with a team model of midwifery care.

This qualitative study investigated midwives' perception of a team midwifery model of care implemented in North Queensland, Australia. A midwifery model of care is the use of primary health care principles to deliver care throughout the woman's entire pregnancy and postpartum period in partnership with other members of the health care team. Four focus groups were undertaken with 22 midwives to determine their perception of the team midwifery model of care. The study found the experience of the team midwifery model of care for midwives had been influenced by organisational characteristics, team structures, and accountability. Recommendations from this study include the need for an appropriate environmental scan and implementation of planning process and team building before the introduction of any new model of care, transportability of health care services to any new model of care, and a shared governance to allow midwives to meet both organisational and professional goals.