|
|
Emma Doud has a multidisciplinary background in biochemistry, mass spectrometry, chemical biology, genetics, and computational / statistical data analysis, applied to a wide number of biological problems as a faculty member in the Department of Biochemistry and Molecular Biology at Indiana University School of Medicine. Her graduate work utilized a combination of genetics and small molecule mass spectrometry techniques. As a research associate at Northwestern University, she led collaborative projects focused on quantitation of histone post-translational modifications, the mechanism of small molecule inhibition of GABA-AT and bottom up and top-down global proteomics using advanced mass spectrometry methods. Since joining the Center for Proteome Analysis as Associate Director in 2019, she's optimized sample preparation workflows from diverse samples including brain, muscle, bone, cartilage, cancerous tumors, and cell lines while rapidly expanding work in proteomics bioinformatics and computational biology. She has served as the lead investigator for database-assisted de novo protein sequence analysis using the algorithm PEAKS. In 2023, she received a grant by the organization 100 Voices of Hope to support he independent research program which is focused on using enrichment techniques and mass spectrometry to understand how post-translational modifications, including acylation, oxidation, phosphorylation and glycosylation impact protein-protein interactions. She's worked closely with the Model-AD team over the last year to create a high throughput assay to quantify the biotherapeutic aducanumab in 5XFAD model mice cortex and plasma samples. This work will be presented at several conferences this summer and published soon. She's used Skyline off and on at Northwestern (targeted histone modification mass spec), in industry (Catalent - oxidation states of therapeutic monoclonal antibodies), and now back in academics in a core facility when targeted mass spectrometry is requested.
|
Although pharmacokinetics and pharmacodynamics of biotherapeutics are commonly studied through ELISAs, the extremely strong binding of modern antibody-based therapeutics can result in background, inability of secondary antibody binding, and nonlinear response curves. The selectivity and specificity of liquid chromatography-targeted mass spectrometry (LC-MS/MS) allows for absolute quantitation of chosen peptides. The Model Organism Development and Evaluation for Late-onset Alzheimer’s Disease (MODEL-AD) Consortium was established to generate, characterize, and validate the next generation of mouse models of AD and to develop a preclinical testing pipeline. As a template workflow for MODEL-AD, we have developed a high-throughput workflow for absolute quantification of chimeric aducanumab from cortex and plasma of 5XFAD mice.
Read More
Methods
A targeted MS assay for quantitation of aducanumab was designed utilizing guidelines described by the National Cancer Institute’s Clinical Proteomic Tumor Analysis Consortium (CPTAC). Proteotryptic peptides unique for chimeric aducanumab were selected, and stable isotope versions were purchased as spike in controls. A high sensitivity and high-throughput methodology was developed for increased sensitivity. Major steps that were optimized using an AssayMap Bravo (Agilent) include: automated Protein A enrichment, reduction, alkylation, trypsin digestion, and loading on to Evotips. An Evosep One LC was paired with a Lumos Tribrid Orbitrap (Thermo Fisher Scientific) for data acquisition and data were analyzed in Skyline (MacCoss lab) with a concentration curve of pure protein in matrix normalized to spike-in stable isotope labeled peptides.
Preliminary Data
The three tryptic peptides used for quantitation of aducanumab had lower limits of detection (LLOD) and quantification of 1-500 amol pure peptide on column, 2-5 ng aducanumab / µL in plasma, and 0.225 ng / µg brain homogenate. Using protein A purification of the biotherapeutic, the lower limit of quantification (LLOQ) in cortex samples was decreased by 100-fold. This assay was sensitive and linear with high reproducibility (CV 3-10% in technical replicates run on separate days) and was micronized for 96 sample formats, where a single plate could be analyzed in 48-72 hours. In the study, a balanced number of male and female 5XFAD or WT mice were dosed with daily 30 mg/kg, weekly 30 mg/kg or weekly 1 mg/kg aducanumab or weekly 30 mg/kg control IgG. A pilot cohort consisted of 37 cortex samples and 222 (37 animals with 6 timepoints) was used for workflow optimization. Following this, blinded samples including 126 cortex and 380 (2 timepoints) plasma samples were used to optimize the workflow. All data and methods will be uploaded to Panorama.
Novel Aspect
A high throughput targeted mass spectrometry workflow to be used for biotherapeutics with the MODEL-AD consortium.
Read Less
