Benzotriazole-Assisted Solid-Phase Assembly of Leu-Enkephalin, Amyloid β segment 34−42, and other “Difficult” Peptide Sequences

2009 ◽  
Vol 74 (5) ◽  
pp. 2028-2032 ◽  
Author(s):  
Alan R. Katritzky ◽  
Danniebelle N. Haase ◽  
Jodie V. Johnson ◽  
Alfred Chung
Keyword(s):  
Solid Phase ◽  
Amyloid Β ◽  
Difficult Peptide Sequences

scholarly journals Peptidomimetic Modulators of BACE1

2020 ◽  
Vol 73 (4) ◽  
pp. 366
Author(s):  
John Paul Juliano ◽  
David H. Small ◽  
Marie-Isabel Aguilar
Keyword(s):  
Amino Acids ◽  
Solid Phase ◽  
Solid Phase Peptide Synthesis ◽  
Amyloid Β ◽  
Amino Acid Sequences ◽  
Peptide Sequence ◽  
Fluorescent Substrate ◽  
Double Mutation ◽  
Affinity Peptide ◽  
Inhibition Potency

The β-site APP Cleaving enzyme 1 (BACE1) is a membrane-associated aspartyl protease which mediates the production of amyloid-β (Aβ), a major component of amyloid plaques in the Alzheimer’s disease brain. We have synthesised and characterised a series of peptidomimetic analogues of BACE substrates that incorporate two distinct stabilising structures. To demonstrate the potential activity of these compounds, a variety of assaying strategies were used to investigate cleavage susceptibility and inhibition potency under competitive and non-competitive conditions. β-Amino acids and scissile site N-methylation were incorporated into peptide substrate templates as transition state isostere (TSI) substitutes by positional scanning to generate series of non-TSI β-peptidomimetics. The amino acid sequences flanking the β-cleavage site within APP carrying the Swedish double mutation (APPSW), Neuregulin, the synthetic hydroxyethylene-based TSI peptide inhibitor OM99-2, and the high affinity peptide sequence SEISYEVEFR, served as the four substrate templates from which over 60 peptides were designed and synthesised by solid phase peptide synthesis. A quenched fluorescent substrate BACE1 assay in conjunction with liquid chromatography–mass spectrometry (LC-MS) analysis was established to investigate cleavage susceptibility and inhibition potency under competitive and non-competitive conditions. It was determined that β-amino acids substituted at the P1 scissile site position within known peptide substrates were resistant to proteolysis, and particular substitutions induced a concentration-dependent stimulation of BACE1, indicating a possible modulatory role of native BACE1 substrates.

scholarly journals Mass Spectrometry–Based Candidate Reference Measurement Procedure for Quantification of Amyloid-β in Cerebrospinal Fluid

2014 ◽  
Vol 60 (7) ◽  
pp. 987-994 ◽  
Author(s):  
Andreas Leinenbach ◽  
Josef Pannee ◽  
Thomas Dülffer ◽  
Andreas Huber ◽  
Tobias Bittner ◽  
...  
Keyword(s):  
Cerebrospinal Fluid ◽  
Solid Phase ◽  
Amyloid Β ◽  
Measurement Procedure ◽  
Matrix Interference ◽  
Expanded Uncertainty ◽  
Reference Measurement ◽  
Reference Measurement Procedure ◽  
Csf Proteins ◽  
Cutoff Levels

Abstract BACKGROUND Cerebrospinal fluid (CSF) amyloid-β (Aβ42) is a well-established biomarker for Alzheimer disease. Several immunoassays for Aβ42 exist but differ in absolute concentrations and may suffer from matrix interference, thereby hampering interlaboratory comparisons and the use of general cutoff levels. Together with the IFCC Working Group on CSF Proteins, we developed a candidate reference measurement procedure (RMP) for Aβ42. METHODS The antibody-independent candidate RMP was based on solid-phase extraction and isotope-dilution LC-MS/MS. The candidate RMP used 2 differently stable isotope-labeled Aβ42 peptides for calibration in human CSF, an important aspect since there was no analyte-free matrix available. Because no CSF certified reference material (CRM) exists, we used a nonlabeled Aβ42 standard, the concentration of which was determined by amino acid analysis. We performed measurements on a high-resolution quadrupole-Orbitrap hybrid instrument. The results were compared with a method run in a second laboratory with triple quadrupole instrumentation. RESULTS The candidate RMP allowed quantification of CSF Aβ42 from 150 to 4000 pg/mL. Validation of the method showed a recovery of 100% (15%), intraassay and interassay imprecision of 5.0% and 6.4%, respectively, and an expanded uncertainty of 15.7%. No analytical interferences or carryover were detected. CONCLUSIONS This method will help set the value of CSF Aβ42 in a CRM, which could be used to harmonize Aβ42 assays and facilitate the introduction of general cutoff concentrations for CSF Aβ42 in clinical trials and practice.

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

ChemInform ◽  
2005 ◽  
Vol 36 (23) ◽  
Author(s):  
Anna K. Tickler ◽  
Andrew B. Clippingdale ◽  
John D. Wade
Keyword(s):  
Peptide Synthesis ◽  
Solid Phase ◽  
Solid Phase Peptide Synthesis ◽  
Amyloid Β ◽  
Difficult Sequence

scholarly journals Solid phase synthesis of four analogs of amyloid-β(9-16) peptide: MS and FT-IR characterization

2019 ◽  
Vol 64 (5) ◽  
pp. 433-443
Author(s):  
Monica JURESCHI ◽  
◽  
Ionel HUMELNICU ◽  
Brîndușa Alina PETRE ◽  
Cătălina-Ionica CIOBANU ◽  
...  
Keyword(s):  
Solid Phase Synthesis ◽  
Solid Phase ◽  
Amyloid Β ◽  
Phase Synthesis ◽  
Ft Ir

scholarly journals Establishing pre-analytical requirements and maximizing peptide recovery in the analytical phase for mass spectrometric quantification of amyloid-β peptides 1–42 and 1–40 in CSF

2021 ◽  
Vol 0 (0) ◽  
Author(s):  
Lauren M. Forgrave ◽  
J. Grace van der Gugten ◽  
Quyen Nguyen ◽  
Mari L. DeMarco
Keyword(s):  
Mass Spectrometry ◽  
Clinical Laboratory ◽  
Comprehensive Evaluation ◽  
Solid Phase ◽  
Amyloid Β ◽  
Careful Consideration ◽  
Mass Spectrometric ◽  
Analytical Sensitivity ◽  
Surrogate Matrix ◽  
Analytical Phase

Abstract Objectives Amyloid-β (Aβ) peptides in cerebrospinal fluid (CSF), including Aβ42 (residues 1–42) and Aβ40 (residues 1–40), are utilized as biomarkers in the diagnostic workup of Alzheimer’s disease. Careful consideration has been given to the pre-analytical and analytical factors associated with measurement of these peptides via immunoassays; however, far less information is available for mass spectrometric methods. As such, we performed a comprehensive evaluation of pre-analytical and analytical factors specific to Aβ quantification using mass spectrometry. Methods Using our quantitative mass spectrometry assay for Aβ42 and Aβ40 in CSF, we investigated the potential for interference from hemolysate, bilirubin, lipids, and anti-Aβ-antibodies. We also optimized the composition of the calibrator surrogate matrix and Aβ recovery during and after solid phase extraction (SPE). Results There was no interreference observed with total protein up to 12 g/L, hemolysate up to 10% (v/v), bilirubin up to 0.5% (v/v), intralipid up to 1% (v/v), or anti-Aβ-antibodies at expected therapeutic concentrations. For hemolysate, bilirubin and lipids, visual CSF contamination thresholds were established. In the analytical phase, Aβ recovery was increased by ∼50% via SPE solvent modifications and by over 150% via modification of the SPE collection plate, which also extended analyte stability in the autosampler. Conclusions Attention to mass spectrometric-specific pre-analytical and analytical considerations improved analytical sensitivity and reproducibility, as well as, established CSF specimen acceptance and rejection criteria for use by the clinical laboratory.

scholarly journals Aluminium Binding to Modified Amyloid-β Peptides: Implications for Alzheimer’s Disease

Molecules ◽  
2020 ◽  
Vol 25 (19) ◽  
pp. 4536
Author(s):  
Cosmin Stefan Mocanu ◽  
Monica Jureschi ◽  
Gabi Drochioiu
Keyword(s):  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
Solid Phase ◽  
Solid Phase Peptide Synthesis ◽  
Amyloid Β ◽  
Molecular Ions ◽  
Amino Acid Residues ◽  
Peptide Fragment ◽  
Peptide Fragments ◽  
Modified Peptides

Aluminium (Al) is clearly neurotoxic and considerable evidence exists that Al may play a role in the aetiology or pathogenesis of Alzheimer’s disease (AD). Nevertheless, the link between AD pathology and Al is still open to debate. Therefore, we investigated here the interaction of aluminium ions with two Aβ peptide fragments and their analogues. First, we synthesised by the Fmoc/tBu solid-phase peptide synthesis (SPPS) strategy using an automated peptide synthesiser two new peptides starting from the Aβ(1–16) native peptide fragment. For this purpose, the three histidine residues (H6, H13, and H14) of the Aβ(1–16) peptide were replaced by three alanine and three serine residues to form the modified peptides Aβ(1–16)A36,13,14 and Aβ(1–16)S36,13,14 (primary structures: H-1DAEFRADSGYEVAAQK16-NH2 and H-1DAEFRSDSGYEVSSQK16-NH2). In addition, the Aβ(9–16) peptide fragment (H-9GYEVHHQK16-NH2) and its glycine analogues, namely Aβ(9–16)G110, (H-9GGEVHHQK16-NH2), Aβ(9–16)G213,14 (H-9GYEVGGQK16-NH2), and Aβ(9–16)G310,13,14 (H-9GGEVGGQK16-NH2), were manually synthesised in order to study Al binding to more specific amino acid residues. Both the peptides and the corresponding complexes with aluminium were comparatively investigated by mass spectrometry (MS), circular dichroism spectroscopy (CD), atomic force microscopy (AFM), scanning electron microscopy (SEM), and Fourier transform infrared spectroscopy (FT-IR). Al–peptide molecular ions and Al-fragment ions were unambiguously identified in the MS and MS/MS spectra. AFM images showed dramatic changes in the film morphology of peptides upon Al binding. Our findings from the investigation of N-terminal 1-16 and even 9-16 normal and modified sequences of Aβ peptides suggest that they have the capability to be involved in aluminium ion binding associated with AD.

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