Dataset Information

The synthesis and characterization of Bri2 BRICHOS coated magnetic particles and their application to protein fishing: Identification of novel binding proteins.

ABSTRACT: Human integral membrane protein 2B (ITM2B or Bri2) is a member of the BRICHOS family, proteins that efficiently prevent Aβ42 aggregation via a unique mechanism. The identification of novel Bri2 BRICHOS client proteins could help elucidate signaling pathways and determine novel targets to prevent or cure amyloid diseases. To identify Bri2 BRICHOS interacting partners, we carried out a 'protein fishing' experiment using recombinant human (rh) Bri2 BRICHOS-coated magnetic particles, which exhibit essentially identical ability to inhibit Aβ42 fibril formation as free rh Bri2 BRICHOS, in combination with proteomic analysis on homogenates of SH-SY5Y cells. We identified 70 proteins that had more significant interactions with rh Bri2 BRICHOS relative to the corresponding control particles. Three previously identified Bri2 BRICHOS interacting proteins were also identified in our 'fishing' experiments. The binding affinity of Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), the top 'hit', was calculated and was identified as a strong interacting partner. Enrichment analysis of the retained proteins identified three biological pathways: Rho GTPase, heat stress response and pyruvate, cysteine and methionine metabolism.

SUBMITTER: Tigro H

PROVIDER: S-EPMC10644258 | biostudies-literature | 2021 May

REPOSITORIES: biostudies-literature

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The synthesis and characterization of Bri2 BRICHOS coated magnetic particles and their application to protein fishing: Identification of novel binding proteins.

Tigro Helene H Kronqvist Nina N Abelein Axel A Galan-Acosta Lorena L Chen Gefei G Landreh Michael M Lyashkov Alexey A Aon Miguel A MA Ferrucci Luigi L Shimmo Ruth R Johansson Jan J Moaddel Ruin R

Journal of pharmaceutical and biomedical analysis 20210226

Human integral membrane protein 2B (ITM2B or Bri2) is a member of the BRICHOS family, proteins that efficiently prevent Aβ42 aggregation via a unique mechanism. The identification of novel Bri2 BRICHOS client proteins could help elucidate signaling pathways and determine novel targets to prevent or cure amyloid diseases. To identify Bri2 BRICHOS interacting partners, we carried out a 'protein fishing' experiment using recombinant human (rh) Bri2 BRICHOS-coated magnetic particles, which exhibit e ...[more]

PMID: 33690096

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Project description:Molecular diagnostic methods to detect and quantify viral RNA in clinical samples rely on the purification of the genetic material prior to reverse transcription polymerase chain reaction (qRT-PCR). Due to the large number of samples processed in clinical laboratories, automation has become a necessity in order to increase method processivity and maximize throughput per unit of time. An attractive option for isolating viral RNA is based on the magnetic solid phase separation procedure (MSPS) using magnetic microparticles. This method offers the advantage over other alternative methods of making it possible to automate the process. In this study, we report the results of the MSPS method based on magnetic microparticles obtained by a simple synthesis process, to purify RNA from oro- and nasopharyngeal swab samples of patients suspected of COVID-19 provided by three diagnostic laboratories located in the Buenos Aires Province, Argentina. Magnetite nanoparticles of Fe3O4 (MNPs) were synthesized by the coprecipitation method and then coated with silica (SiO2) produced by hydrolysis of tetraethyl orthosilicate (TEOS). After preliminary tests on samples from the A549 human lung cell line and swabs, an extraction protocol was developed. The quantity and purity of the RNA obtained were determined by gel electrophoresis, spectrophotometry, and qRT-PCR. Tests on samples from naso- and oropharyngeal swabs were performed in order to validate the method for RNA purification in high-throughput SARS-CoV-2 diagnosis by qRT-PCR. The method was compared to the spin columns method and the automated method using commercial magnetic particles. The results show that the method developed is efficient for RNA extraction from nasal and oropharyngeal swab samples, and also comparable to other extraction methods in terms of sensitivity for SARS-CoV-2 detection. Of note, this procedure and reagents developed locally were intended to overcome the shortage of imported diagnostic supplies as the sudden spread of COVID-19 required unexpected quantities of nucleic acid isolation and diagnostic kits worldwide.

Dataset Information

The synthesis and characterization of Bri2 BRICHOS coated magnetic particles and their application to protein fishing: Identification of novel binding proteins.

Publications

The synthesis and characterization of Bri2 BRICHOS coated magnetic particles and their application to protein fishing: Identification of novel binding proteins.

Similar Datasets

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