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Identification of SVEP1 protein interactions using affinity and proximity-based purifications coupled with mass spectrometry

ABSTRACT: SVEP1 is a putative extracellular matrix (ECM) protein that promotes atherosclerosis in humans and mice. Antibodies that reliably recognize SVEP1 in situ have not been successfully developed. As a result, little is known about how and where SVEP1 may integrate into the ECM or which cell types it may influence. We used a combination of affinity and proximity-based experimental approaches to address this question. The bait proteins for these experiments included recombinant murine SVEP1 fused to a Myc-tag to allow for affinity-based co-immunoprecipitation or the promiscuous biotin ligase mini-Turbo ID to allow for proximity-based labeling and pulldown with streptavidin beads. The prey proteins were derived from enriched media of cultured murine VSMCs. Two independent experiments were performed using each approach and a reproducibility criterion of P < 0.10 for enrichment was applied across all experimental data. In addition to SVEP1, other ECM proteins that were identified include Basement membrane-specific heparan sulfate proteoglycan core protein (HSPG2, also known as Perlecan), Fibronectin, Laminin subunit gamma-1, and Nidogen-1. Together, these proteins comprise the major non-collagen basement membrane components. This suggests SVEP1 may be integrated into the basement membrane.

INSTRUMENT(S): timsTOF Pro, Q Exactive Plus

ORGANISM(S): Mus Musculus (ncbitaxon:10090)

SUBMITTER: Nathan Stitziel MD, PhD

PROVIDER: MSV000090134 | MassIVE | Mon Aug 15 07:50:00 BST 2022

SECONDARY ACCESSION(S): PXD036060

REPOSITORIES: MassIVE

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