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Proteomics of isolated human cerebral vessels and whole tissue

ABSTRACT: Cerebral vasculature is critical in maintaining brain health, as its dysfunction is linked to various neurological illnesses including dementia and stroke. Proteomic studies are integral in understanding the biological mechanism underlying this dysfunction, identifying potential therapeutic targets and novel biomarkers of disease. However, translation of research findings into clinical practice relies on the validity and reproducibility of its methods. Thus, we have developed a novel protocol for the extraction of human cerebral vasculature which is highly compatible with downstream proteomic analyses. This protocol negates the need for bovine serum albumin, a ubiquitous reagent in vessel isolation protocols, yet an undesirable contaminant in proteomics.

INSTRUMENT(S):

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Brain

SUBMITTER: Blanca Díaz Castro

LAB HEAD: Blanca Diaz-Castro

PROVIDER: PXD055837 | Pride | 2025-09-11

REPOSITORIES: Pride

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Project description:Severe hyposalivation often results from Sjögren’s syndrome or radiation therapy for head and neck cancer. Devastating consequences of hyposalivation, such as rampant dental caries and persistent oral candidiasis, compromise the quality of life in those patients. Clinical management for dry mouth typically involves simple palliative methods or secretagogues, which are designed to stimulate saliva secretion from residual salivary acinar cells present in the glands. However, direct interventions in chronic dryness have yet to be employed in the clinical setting. Despite numerous studies on salivary gland regeneration, the molecular basis governing stem cell transdifferentiation into salivary epithelial precursors (SEP) is largely unknown. Our previously published study clearly indicated mouse bone marrow-derived mesenchymal stem cells (BM-MSCs) can be differentiated into SEP in vitro when co-cultured with isolated primary salivary gland cells without cell-to-cell contact. Our current study utilized iTRAQ-LS-MS/MS-based quantitative proteomics to profile key regulatory factors involved in mouse MSC-to-SEP conversion. We identified 280 differentially expressed proteins in BM-MSCs over the course of 7 days of co-culture. Interestingly, protein expression of salivary transcription factors (STFs), such as transcription factor E2a (TCF3), high mobility group protein 20B (HMG20b), and ankyrin repeat domain-containing protein 56 (ANKRD56) were increased in a time-dependent manner. Notably, pancreas specific transcription factor 1a (PTF1α), muscle, intestine and stomach expression-1 (MIST-1) and achaete-scute complex homolog 3(ASCL3) were newly induced over time in differentiated MSCs. We also identified the expression of Ptf1α in the mouse salivary glands for the first time and verified its expression in independent batches of co-cultured MSCs by western blotting. Furthermore, simulation of the molecular network involving the identified STFs has demonstrated evidence for their perspective roles in salivary gland development during glandular maturation. Thus, our study provides the first extensive proteomic profile of MSC-to-SEP transdifferentiation and identifies novel STFs that may be critical for this process.

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Proteomics of isolated human cerebral vessels and whole tissue

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