Amyloid‐β as a “Difficult Sequence” in Solid Phase Peptide Synthesis

Tickler, Anna K.; Clippingdale, Andrew B.; Wade, John D.

doi:10.1002/chin.200523256

Cited by 12 publications

(16 citation statements)

References 1 publication

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“…Complementary techniques such as SDS-PAGE, size exclusion chromatography, and/or dynamic light scattering (DLS) also report on the quality of the starting material. While 95% purity is the minimal acceptable level, common contaminants rise from truncated sequences (peptides missing one or more amino acids) [53][54][55], amino acid racemization, side reactions during the peptide cleavage, and deprotection and oxidation of amino acids (in particular methionine (Met)), which occur during the synthesis. Met oxidation can be minimized with proper synthesis methodology [55,56]).…”

Section: Purity Of Peptidesmentioning

confidence: 99%

ThT 101: a primer on the use of thioflavin T to investigate amyloid formation

Malmos

Blancas‐Mejia

Weber

et al. 2017

Amyloid

301

243

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Thioflavin T (ThT) has been widely used to investigate amyloid formation since 1989. While concerns have recently been raised about its use as a probe specific for amyloid, ThT still continues to be a very valuable tool for studying kinetic aspects of fibrillation and associated inhibition mechanisms. This review aims to provide a conceptual instruction manual, covering appropriate considerations and pitfalls related to the use of ThT. We start by giving a brief introduction to amyloid formation with focus on the morphology of different aggregate species, followed by a discussion of the quality of protein needed to obtain reliable fibrillation data. After an overview of the photochemical basis for ThT's amyloid binding properties and artifacts that may arise from this, we describe how to plan and analyze ThT assays. We conclude with recommendations for complementary techniques to address shortcomings in the ThT assay.

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Section: Purity Of Peptidesmentioning

confidence: 99%

ThT 101: a primer on the use of thioflavin T to investigate amyloid formation

Malmos

Blancas‐Mejia

Weber

et al. 2017

Amyloid

301

243

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“…Synthesis of the human β-amyloid (1-42) peptide (H-DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMV-GGVVIA-OH) by conventional SPPS has been reported to be difficult owing to the high hydrophobicity of the C-terminal segment and on-resin aggregation [18,19]. We initially synthesized β-amyloid 1 -42 with deprotection times of 15 min × 1, acylation times of 35 min, and capping times of 5 min for a total synthesis time of 47.8 h. β-Amyloid 1 -42 was then synthesized with 1 min × 2 deprotection 5 min acylation times, and 2.5 min × 2 capping times giving a total synthesis time of 14 h and produced a crude peptide with similar purity to the long synthesis.…”

Section: β-Amyloid (1-42)mentioning

confidence: 99%

Fast conventional Fmoc solid‐phase peptide synthesis with HCTU

Hood

Fuentes

Patel

et al. 2007

Journal of Peptide Science

144

121

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1H-Benzotriazolium 1-[bis(dimethyl-amino)methylene]-5-chloro-hexafluorophosphate (1-),3-oxide (HCTU) is a nontoxic, nonirritating and noncorrosive coupling reagent. Seven biologically active peptides (GHRP-6, 65 -74 ACP, oxytocin, G-LHRH, Cpeptide, hAmylin 1 -37 , and β-amyloid 1 -42 ) were synthesized with reaction times reduced to deprotection times of 3 min or less and coupling times of 5 min or less using HCTU as the coupling reagent. Expensive coupling reagents or special techniques were not used. Total peptide synthesis times were dramatically reduced by as much as 42.5 h (1.8 days) without reducing the crude peptide purities. It was shown that HCTU can be used as an affordable, efficient coupling reagent for fast Fmoc solid-phase peptide synthesis.

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“…Several spectroscopic studies have indicated that the copper-binding site of Aβ consists of the three histidine residues His6, His13 and His14 and an as yet undefined fourth ligand; proposed ligands include Tyr10 [19,[38][39][40], the Nterminal nitrogen [30] or an as yet unidentified carboxylate side chain [31,32].…”

Section: Accepted M Manuscriptmentioning

confidence: 99%

Interaction of β-amyloid(1-40) peptide with pairs of metal ions: An electrospray ion trap mass spectrometric model study

Drochioiu

Manea

Drăguşanu

et al. 2009

Biophysical Chemistry

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This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT 2 AbstractThe stoichiometries and the affinity toward simple and paired metal ions of synthetic amyloid-β(1-40)peptide (Aβ1-40) were investigated by electrospray ion trap mass spectrometry (ESI-MS), circular dichroism (CD), and atomic force microscopy (AFM). The results lead to the working hypothesis that pHdependent metal binding to Aβ1-40 may induce conformational changes, which affect the affinity toward other metals. A significant copper and zinc binding to Aβ1-40 peptide at pH 5.5 was found, whereas nickel ions commonly bind to each molecule of β-amyloid peptide. Some complexes of Aβ1-40 with more than one nickel ion were identified by ESI-MS. In addition, nickel ions proved to enhance Aβ oligomerization. On increasing pH, up to 12 ions of zinc may bind to a single Aβ molecule. Under the same pH and concentration conditions, the binding pattern of the independent copper and silver ions to Aβ1-40 was different from that of the equimolecular mixture of the two metal ions. One might assume that some conformational changes due to water loss altered the capacity of Aβ peptide to bind certain heavy metal ions. As a consequence, copper-silver interaction with the binding process to Aβ1-40 became highly complex. A competition between silver and nickel ions for Aβ1-40 binding sites at high pH was also observed. New strategies were proposed to identify the characteristic signals for some important metal ion-peptide complexes in the spectra recorded at high pH or high concentrations of metal ions. To explain the formation of such a large number of high metal ion-Aβ complexes, we took into consideration the participation of both histidine residues and free amino groups as well as carboxylate ones in the binding process. Finally, CD and AFM studies supported the mass spectrometric data.

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Amyloid‐β as a “Difficult Sequence” in Solid Phase Peptide Synthesis

Abstract: For Abstract see ChemInform Abstract in Full Text.

Cited by 12 publications

References 1 publication

ThT 101: a primer on the use of thioflavin T to investigate amyloid formation

ThT 101: a primer on the use of thioflavin T to investigate amyloid formation

Fast conventional Fmoc solid‐phase peptide synthesis with HCTU

Interaction of β-amyloid(1-40) peptide with pairs of metal ions: An electrospray ion trap mass spectrometric model study

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