DOI: 10.48349/asu/ztw1hn
title: Replication data for: Uncovering temporospatial sensitive TBI targeting strategies via in vivo phage display
title: In vivo phage display DNA sequences and mass spectrometry data

description: &lt;p&gt;The heterogeneous pathophysiology of traumatic brain injury (TBI) is a barrier to advancing diagnostics and therapeutics, including targeted drug delivery. We employed a unique discovery pipeline to identify novel targeting motifs that recognize specific temporal phases of TBI pathology. This pipeline combined in vivo biopanning with domain antibody (dAb) phage display, next-generation sequencing analysis, and peptide synthesis. We identified targeting motifs based on the complementarity-determining region 3 structure of dAbs for acute (1 day post-injury) and subacute (7 days post-injury) post-injury timepoints in a pre-clinical TBI model (controlled cortical impact; CCI). Bioreactivity and temporal sensitivity of the targeting motifs were validated via immunohistochemistry. Immunoprecipitation-mass spectrometry indicated that the acute TBI targeting motif recognized targets associated with metabolic and mitochondrial dysfunction whereas the subacute TBI motif was largely associated with neurodegenerative processes. This pipeline successfully discovered temporally specific TBI targeting motif/epitope pairs that will serve as the foundation for the next generation targeted TBI therapeutics and diagnostics.&lt;/p&gt;

&lt;p&gt;&lt;em&gt;Methods used for collection/generation of data&lt;/em&gt;&lt;/p&gt;
&lt;p&gt;IMMUNOPRECIPITATION-MASS SPECTROMETRY:&lt;/p&gt;
&lt;p&gt;CCI and sham surgeries were completed as described previously (n = 3 biological replicates per timepoint). Mice were sacrificed at 1 or 7 dpi via transcardial perfusion with phosphate buffer, pH 7.4. The ipsilateral hemisphere of the brain was immediately dissected and homogenized in chilled lysis buffer (1X PBS, 1% Triton, protease inhibitor cocktail). Protein concentration was quantified with the Pierce BCA Protein Assay Kit (Thermo Fisher)&lt;/p&gt;
&lt;p&gt;Streptavidin-coupled Dynabeads (Thermo Fisher) were washed with 0.1% Tween in 1X PBS and incubated with 1 mg/mL tissue lysate for 1 hour at room temperature. Pre-cleared lysate was collected after separation from magnetic beads and incubated with designated HCDR3-constructs rotating overnight at 4°C to form the immune complex. The immune complex was incubated with streptavidin-coupled Dynabeads for 1 hour at room temperature and beads were then washed 3 times with chilled lysis buffer. Antigens were eluted directly from beads with 0.2% Rapigest for LC-MS analysis using the Thermo Orbitrap Fusion Lumos (Thermo Fisher) by the ASU Biodesign Mass Spectrometry Facility.&lt;/p&gt;

&lt;p&gt;&lt;em&gt;Methods for processing the data:&lt;/em&gt;&lt;/p&gt;
&lt;p&gt;IP-MS data were collected using the Thermo Fisher Thermo Orbitrap Fusion Lumos.2.4.1.15&lt;/p&gt;

&lt;p&gt;&lt;em&gt;Environmental/experimental conditions:&lt;/em&gt;&lt;/p&gt;
&lt;p&gt;All experiments were approved by the Arizona State University Institutional Animal Care and Use Committee (IACUC) under protocols #17-1590R and #20-1793R. 
Eight-week-old male and female C57Bl/6 mice (Charles River) were assigned to four experimental groups; acute (1 dpi), subacute (7 dpi), chronic (21 dpi) and sham (craniotomy with no injury, sacrificed 1-day post-procedure). &lt;/p&gt;
&lt;p&gt;&lt;p&gt;See &lt;a href="https://dataverse.asu.edu/file.xhtml?fileId=9843"&gt;README&lt;/a&gt; for additional methodological information on data collection, data processing, experimental conditions, and abbreviations.&lt;/p&gt;
description: Thermo Fisher Thermo Orbitrap Fusion Lumos, 2.4.1.15