Ashley Frankenfield, Graduate Student, Hao Lab, GW Department of Chemistry

The Department of Chemistry Presents:  Ashley Frankenfield, Graduate Student, Hao Lab, GW Department of Chemistry

Neurons are postmitotic cells that rely heavily on the lysosome autophagy pathway to prevent the accumulation of misfolded and aggregated proteins within the brain. However, which proteins interact with the lysosome and how turnover occurs in healthy neurons is not well understood. Here, we first developed and systematically optimized a proximity-based labeling method that captured the stable and transient protein-protein interactions occurring along the lysosome membrane. Quantification accuracy in proximity-labeling experiments is often limited by the presence of abundant background interference from experimental protein reagents added throughout the sample preparation methods. To address this problem, we developed a contaminant spectral library that can readily be integrated into all proteomics data analysis software and has been shown to reduce false discoveries.

Furthermore, we systematically improved a mass spectrometry-based dynamic SILAC proteomic approach to enable a deep and accurate measurement of protein turnover in human induced pluripotent stem cell (iPSC)-derived motor and cortical neurons. We benchmarked common DDA and DIA data analysis platforms for SILAC proteomics to compare the accuracy of each tool. All samples were then extensively fractionated to increase neuronal proteome coverage, allowing us to calculate the turnover of 10,372 proteins and 148,943 peptides in human iPSC-derived neurons. We discovered that proteins involved in synaptic transmission, structure and metabolic processes have significantly different half-lives in motor and cortical neurons. Using this comprehensive study, we created NeuronProfile (www.neuronprofile.com), an interactive website for visualizing and querying protein turnover in subcellular locations in human neurons. Furthermore, we applied this method to evaluate how common cellular stresses (i.e., genetic mutation, nutrient starvation, drug treatment) impact protein turnover.

BIO

Ashley received her B.S. in chemical biology and B.B.A. in finance from Saint Joseph’s University in Philadelphia, PA. She was an undergraduate researcher in Dr. Jose Cerda’s lab studying the thermodynamic constants for fluoride binding to heme-cofactors. Ashley then joined the Chemistry Department at the George Washington University under Dr. Ling Hao. Her current work focuses on developing mass spectrometry techniques for proteomics.