Ashley Frankenfield, Graduate Student, Hao Lab & Sara Mattson, Graduate Student,Rodriguez Lab, Graduate Students, GW Department of Chemistry

Fri, 12 November, 2021 2:00pm

The Department of Chemistry Presents, via Online and In-person Presentation--

Development of a Mass Spectrometry-based Proteomics Method to Study the Lysosome Microenvironment

Proximity-based labeling captures stable and transient protein-protein interactions using a biotin ligase or peroxidase, such as ascorbate peroxidase (APEX2). Within this field, interference of high abundant endogenously biotinylated proteins, and experimental variations among biological/technical replicates hinder data with false discoveries. Here, we have demonstrated how to systematically optimize the parameters of proximity labeling mass spectrometry experiments for better data reproducibility and quantification accuracy. Additionally, we have addressed challenges within the field, such as the presence of endogenously biotinylated mitochondrial proteins that are enriched for using a biotin-streptavidin purification system leading to false discoveries.

In this study, we have developed a new analytical tool for investigating the lysosome-autophagy pathway by genetically fusing the APEX2 enzyme to the lysosome-associated membrane protein 1 (LAMP1) of human iPSC-derived neurons. The autophagy pathway maintains cellular homeostasis by facilitating the degradation and recycling of macromolecules during times of starvation. Within neurons, lysosomes play an additional role in the transport of neurotransmitters. We have developed endogenous and overexpression LAMP1-APEX2 probes that allow protein-protein interactions of the lysosome membrane to be studied. Our findings confirmed that our APEX2 probes capture interactions of proteins related to lysosome acidification, transport, mobility and regulatory pathways.

Bio:

Ashley received her bachelor’s degree in chemical biology from Saint Joseph’s University in Philadelphia, PA. In fall 2019, she began her PhD in Dr. Ling Hao’s lab at George Washington University. Ashley's current research focus is on developing and improving proteomic methods to study dynamic protein turnover and lysosomal interactions in human stem cell-derived neurons. She utilizes mass spectrometry and
bioinformatics to identify candidate protein biomarkers in neurodegenerative disorders.”

Single PCR Mammalian Expression Generated Array Directed Evolution of Biomolecules

Directed evolution is a widely used technique for engineering biomolecules to locate and treat disease, remove plastic or carbon dioxide from the environment, develop synthetic biological systems, and synthesize complex molecules without organic solvents. Previous directed evolution methods are time-consuming, limited in biomolecule number and size, and often require hazardous viruses. In this work, I developed a novel directed evolution protocol with a single error prone polymerase chain reaction (PCR) to amplify an entire plasmid containing a fluorescent protein and heme oxygenase-1 to produce biliverdin. The biomolecules are expressed in mammalian and bacterial cells. The novel method removes most molecular biology steps for faster evolution with fewer errors. The evolution of fluorescent proteins that attach biliverdin is necessary in mammalian cells to compete with high affinity, endogenous proteins. High-throughput screening is performed by fluorescence activated cell sorting (FACS) to screen 50 million cells per hour. Using the new method, we created billions of small Ultra-Red Fluorescent Protein (smURFP) variants and selected new variants that are brighter without biliverdin addition. We developed an entirely new class of fluorescent protein from a frog pigmentation protein, which was initially non-fluorescent and now is 40-fold brighter than smURFP in mammalian cells. The patent-pending directed evolution method allows for the evolution of multiple biomolecules inside cells quickly and efficiently, which is fully automatable in biotechnology companies.

Bio:

Sara Mattson received her M.S. in Chemistry from American University in Washington DC. She performed graduate research with Dr. Monika Konaklieva to synthesize electrophilic small molecules to activate lipoprotein lipase to treat heart disease. Ms. Mattson received her B.S. in Chemistry from the State University of New York, Plattsburgh. Sara Mattson joined Professor Erik Rodriguez’s research group in Spring 2021 and developed new directed evolution methods to create new fluorescent proteins for biological applications.