NDLI: The proteome of the blood–brain barrier in rat and mouse: highly specific identification of proteins on the luminal surface of brain microvessels by in vivo glycocapture

Content Provider	Springer Nature : BioMed Central
Author	Tremblay, Tammy-Lynn Alata, Wael Slinn, Jacqueline Baumann, Ewa Delaney, Christie E. Moreno, Maria Haqqani, Arsalan S. Stanimirovic, Danica B. Hill, Jennifer J.
Abstract	Background The active transport of molecules into the brain from blood is regulated by receptors, transporters, and other cell surface proteins that are present on the luminal surface of endothelial cells at the blood–brain barrier (BBB). However, proteomic profiling of proteins present on the luminal endothelial cell surface of the BBB has proven challenging due to difficulty in labelling these proteins in a way that allows efficient purification of these relatively low abundance cell surface proteins. Methods Here we describe a novel perfusion-based labelling workflow: in vivo glycocapture. This workflow relies on the oxidation of glycans present on the luminal vessel surface via perfusion of a mild oxidizing agent, followed by subsequent isolation of glycoproteins by covalent linkage of their oxidized glycans to hydrazide beads. Mass spectrometry-based identification of the isolated proteins enables high-confidence identification of endothelial cell surface proteins in rats and mice. Results Using the developed workflow, 347 proteins were identified from the BBB in rat and 224 proteins in mouse, for a total of 395 proteins in both species combined. These proteins included many proteins with transporter activity (73 proteins), cell adhesion proteins (47 proteins), and transmembrane signal receptors (31 proteins). To identify proteins that are enriched in vessels relative to the entire brain, we established a vessel-enrichment score and showed that proteins with a high vessel-enrichment score are involved in vascular development functions, binding to integrins, and cell adhesion. Using publicly-available single-cell RNAseq data, we show that the proteins identified by in vivo glycocapture were more likely to be detected by scRNAseq in endothelial cells than in any other cell type. Furthermore, nearly 50% of the genes encoding cell-surface proteins that were detected by scRNAseq in endothelial cells were also identified by in vivo glycocapture. Conclusions The proteins identified by in vivo glycocapture in this work represent the most complete and specific profiling of proteins on the luminal BBB surface to date. The identified proteins reflect possible targets for the development of antibodies to improve the crossing of therapeutic proteins into the brain and will contribute to our further understanding of BBB transport mechanisms.
Related Links	https://fluidsbarrierscns.biomedcentral.com/counter/pdf/10.1186/s12987-024-00523-x.pdf
Ending Page	16
Page Count	16
Starting Page	1
File Format	HTM / HTML
ISSN	20458118
DOI	10.1186/s12987-024-00523-x
Journal	Fluids and Barriers of the CNS
Issue Number	1
Volume Number	21
Language	English
Publisher	BioMed Central
Publisher Date	2024-03-04
Access Restriction	Open
Subject Keyword	Neurosciences Hematology Neurobiology Blood–brain barrier Proteomics Luminal Endothelial Vessel
Content Type	Text
Resource Type	Article
Subject	Neurology Developmental Neuroscience Medicine Cellular and Molecular Neuroscience
Journal Impact Factor	5.9/2023
5-Year Journal Impact Factor	7.5/2023

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1	Ministry of Education (GoI), Department of Higher Education	Sanctioning Authority	https://www.education.gov.in/ict-initiatives
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4	Project PI / Joint PI	Principal Investigator and Joint Principal Investigators of the project	Dr. B. Sutradhar bsutra@ndl.gov.in Prof. Saswat Chakrabarti will be added soon
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