33 - Mapping the surface proteome of neonatal hypoxic-ischemic brain injury

Publication Number: 1028.33

Amanda O. Johnson, University of Nebraska College of Medicine, Omaha, NE, United States; Roneldine Mesidor, University of Nebraska College of Medicine, Omaha, NE, United States; Rajnish K. Yadav, University of Nebraska Medical Center, Omaha, NE, United States; Rebekah Gundry, University of Nebraska Medical Center, Omaha, NE, United States; Eric S. Peeples, University of Nebraska Medical Center, Omaha, NE, United States

Background: Neonatal hypoxic-ischemic brain injury (HIBI) is a devastating neurological injury from lack of oxygen and blood flow to the brain, annually affecting up to 26/1000 live births globally. Therapeutic hypothermia is neuroprotective but must be started within 6 hours. Despite this clinical urgency, rapid, accurate bedside diagnostics are currently unavailable. Many infants with HIBI die, and nearly half of survivors suffer severe disability, underscoring a need for additional therapies. Cell surface proteins are drug targets (>60%) and are released during injury, but the HIBI cell surface protein population has never been specifically explored. Our collaborator Dr. Gundry developed CellSurfer, a novel proteomics tool, which we use to map this surfaceome.

Objective: Our objective is to identify and quantify surfaceome changes 30 minutes after HIBI in a mouse model. We hypothesize that HIBI alters the abundance of cell surface proteins that may be a) shed from cells and detectable as circulating injury-specific markers and b) effective targets for neuroprotective drugs.

Design/Methods: Postnatal day 9 mice underwent unilateral carotid artery ligation + 30 min of 8% oxygen or sham surgery + normoxia as controls. Brain cells were dissociated mechanically with collagenase IV, the surface proteins were chemically biotinylated for isolation from the remainder of the cell’s proteome and analyzed via mass spectrometry. CellSurfer tool Veneer accesses Drugbank and predicted drug binding. Bioinformatical validation of protein presence at the surface of the cell were provided with a CIRFESS score and protein cleavage patterns.

Results: So far, with n=3 in each group, we have detected over 900 proteins, roughly 260 of which are present in different abundances between HIBI and sham groups. Proteins unique to the HIBI group include amyloid beta precursor protein (previously documented as upregulated hours after HIBI) and lactoferrin (under investigation for its protective effect after HIBI and potentially of diagnostic interest). Additionally, several drug targets (like brain calcium channel II and Serpin C1) were discovered in higher abundance after HIBI whose modulation may be of therapeutic significance in the context of HIBI by reducing known pathogenic mechanisms from excitotoxicity to ferroptosis.

Conclusion(s): Further work will continue to validate the surfaceome after HIBI with larger group sizes in vivo and in organotypic modelling. Preliminary results demonstrate differences in the surfaceome after HIBI, with promising proteins for diagnostic and therapeutic uses.