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Nanospectral data of amyloid beta 1-42 and scrambled amyloid beta 1-42

ABSTRACT: Two carrier-free peptides were synthesized and used in experiments: amyloid beta 42 (DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA), and a scrambled variant with the same chemical constituency as amyloid beta 42 (AIAEGDSHVLKEGAYMEIFDVQGHVFGGKIFRVVDLGSHNVA). To perform blockade current measurements, first, the sub-nanopore was wetted by immersion in de-gassed 250 mM NaCl electrolyte for 1-3 days. Subsequently, to measure the blockade current, a transmembrane voltage bias (< 700 mV) was applied to the reservoir relative to the ground in the channel using Ag/AgCl electrodes and the corresponding pore current was measured using either an Axopatch 700B or an Axopatch 200B amplifier with an open bandwidth. The actual bandwidth was inferred from the rise-time to a sharp (10 ps rise-time) input pulse to be about 75 kHz to 100 kHz, depending on the amplifier and the feedback. The analog data were digitized by a 16-bit DigiData 1550B data acquisition system (DAQ, Molecular Devices, Sunnyvale, CA) at a sampling rate of 500 kS/s and recorded in 3 minute-long acquisition windows. A total of 12 Axon binary files (ABF) were collected for amyloid beta 42, and 71 ABF files for scrambled amyloid beta 42.

INSTRUMENT(S): Sub-nanopore

ORGANISM(S): Synthesized Peptides

SUBMITTER: Gregory Timp

PROVIDER: MSV000089779 | MassIVE | Thu Jun 30 10:00:00 BST 2022

REPOSITORIES: MassIVE

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Project description:The prevention or delay of brain senescence would enhance the quality of life for older persons. We investigated the effects of soybean extracts in senescence-accelerated (SAMP10) mice. This mouse is a model of brain senescence with a short life span, cerebral atrophy and cognitive dysfunction. Mice were fed a diet containing soybean extracts from 1 to 12 months of age. The effects of green and yellow soybean extracts were compared with a control diet without soybean extracts. Cognitive functions were higher in aged mice fed green soybean than age-matched control mice and mice fed yellow soybean. We further investigated transcriptome of the SAMP10 hippocampus indicated that expression levels of 36 genes were significantly higher and 19 genes were lower in mice that ingested green soybean than in mice that ingested yellow soybean. Some of the evidences were reconfirmed by real time PCR analysis; the levels of Cdh1 and Ptgds mRNA were significantly higher and that the level of Aplp1 was significantly lower in aged SAMP10 mice fed green soybean than mice ingested yellow soybean and control mice. Additionally, the amount of amyloid beta 40 and 42 was lower in the insoluble fraction of aged SAMP10 mice fed green soybean than control mice and mice fed yellow soybean, although the levels of amyloid beta 40 and 42 in the soluble fraction were not different. Lipocalin-type prostaglandin D2 synthase (L-PGDS) has been proposed as the endogenous amyloid beta - chaperone, suggesting that amyloid aggregation was lower in mice fed green soybean than control mice and mice fed yellow soybean. These results indicate that the intake of green soybean improved cognitive function in aged mice, and suppressed amyloid beta accumulation. Green soybean might help healthy aging of the brain in older persons. The effect of green and yellow soybean extracts on cognitive function in aged SAMP10 mice. Mice were fed a CE-2 diet containing 3.0% soybean extracts taken from both yellow and green soybean species, from 1 to 12 months of age. Total RNA was extracted from the stored hippocampus for DNA microarray analysis.

Project description:The amyloid beta (Aβ) peptide represents a 37 to 49 amino acids endoproteolytic fragment of the amyloid precursor protein. The cellular biology that governs the formation and clearance of Aβ has been understood to play a critical role in Alzheimer’s disease (AD). The primary objective of this study was to generate an in-depth inventory of human brain proteins that oligomeric preparations of Aβ1-42 can bind to using an unbiased in vitro discovery approach. Synthetic Aβ1-42 peptides and a brain extract generated from adult human frontal lobe tissue served in these studies as baits and biological source material, respectively. oAβ1-42 was prepared by aggregating the peptide at 4 ºC for 24 h, using previously described procedures known to generate amyloid-β-derived diffusible ligands (ADDLs). Because the interaction with a given binding partner may rely on a binding epitope that comprises N- or C-terminal residues of Aβ1-42, two separate experiments (I and II) were conducted, which differed in the orientation the oAβ1-42 bait was tethered to the affinity matrix. To facilitate meaningful comparisons across experiments, the method of Aβ1-42 capture was not based on immunoaffinity reagents. Instead, alternative Aβ1-42 baits were equipped with biotin moieties attached to the N- or C-terminus by a 6-carbon linker chain, enabling their consistent affinity-capture on streptavidin agarose matrices. Biotin-saturated streptavidin agarose matrices served as negative controls and three biological replicates of samples and controls were generated for each of the three separate interactome datasets by reproducing the affinity-capture step side-by-side on three separate streptavidin agarose affinity matrices that had been saturated with the biotinylated baits. To identify differences in protein-protein interactions of monomeric versus oligomeric Aβ1-42, a 3rd interactome experiment was conducted in which oAβ1-42-biotin or mAβ1-42-biotin served as baits. Digitonin-solubilized brain extracts, which are known to comprise extracellular and most cellular proteins (except for nuclear proteins) served as biological starting material, consistent with the main subcellular areas previously reported to harbor Aβ. Following extensive washes of affinity matrices in their protein-bound state, binders to the bait peptides were eluted by rapid acidification, fully denatured in 9 M urea, and trypsinized. To avoid notorious confounders related to variances in the subsequent handling and analysis of samples, individual peptide mixtures were labeled with distinct isobaric tandem mass tags (TMT) in a six-plex format, then combined and concomitantly subjected to ZipTip-based pre-analysis clean-up by strong cation exchange (SCX) and reversed phase (RP) separation. Four-hour split-free reversed phase nanospray separations were online coupled to an Orbitrap Fusion Tribrid mass spectrometer, which was configured to run an MS3 analysis method.

Project description:INTRODUCTION: The pathogenicity at differing points along the aggregation pathway of many fibril-forming proteins associated with neurodegenerative diseases is unclear. Understanding the effect of different aggregation states of these proteins on cellular processes is essential to enhance understanding of diseases and provide future options for diagnosis and therapeutic intervention.</br>OBJECTIVES:To establish a robust method to probe the metabolic changes of neuronal cells and use it to monitor cellular response to challenge with three amyloidogenic proteins associated with neurodegenerative diseases in different aggregation states.</br>METHOD: Neuroblastoma SH-SY5Y cells were employed to design a robust routine system to perform a statistically rigorous NMR metabolomics study into cellular effects of sub-toxic levels of alpha-synuclein, amyloid-beta 40 and amyloid-beta 42 in monomeric, oligomeric and fibrillar conformations.</br>RESULTS: This investigation developed a rigorous model to monitor intracellular metabolic profiles of neuronal cells through combination of existing methods. This model revealed eight key metabolites that are altered when neuroblastoma cells are challenged with proteins in different aggregation states. Metabolic pathways associated with lipid metabolism, neurotransmission and adaptation to oxidative stress and inflammation are the predominant contributors to the cellular variance and intracellular metabolite levels. The observed metabolite changes for monomer and oligomer challenge may represent cellular effort to counteract the pathogenicity of the challenge, whereas fibrillar challenge is indicative of system shutdown. This implies that although markers of stress are more prevalent under oligomeric challenge the fibrillar response suggests a more toxic environment.</br>CONCLUSION: This approach is applicable to any cell type that can be cultured in a laboratory (primary or cell line) as a method of investigating how protein challenge affects signalling pathways, providing additional understanding as to the role of protein aggregation in neurodegenerative disease initiation and progression.

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Nanospectral data of amyloid beta 1-42 and scrambled amyloid beta 1-42

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