Amyloid β‑Protein Assembly: Differential Effects of the Protective A2T Mutation and Recessive A2V Familial Alzheimer’s Disease Mutation

Oligomeric states of the amyloid β-protein (Aβ) appear to be causally related to Alzheimer’s disease (AD). Recently, two familial mutations in the amyloid precursor protein gene have been described, both resulting in amino acid substitutions at Ala2 (A2) within Aβ. An A2V mutation causes autosomal recessive early onset AD. Interestingly, heterozygotes enjoy some protection against development of the disease. An A2T substitution protects against AD and age-related cognitive decline in non-AD patients. Here, we use ion mobility-mass spectrometry (IM-MS) to examine the effects of these mutations on Aβ assembly. These studies reveal different assembly pathways for early oligomer formation for each peptide. A2T Aβ42 formed dimers, tetramers, and hexamers, but dodecamer formation was inhibited. In contrast, no significant effects on Aβ40 assembly were observed. A2V Aβ42 also formed dimers, tetramers, and hexamers, but it did not form dodecamers. However, A2V Aβ42 formed trimers, unlike A2T or wild-type (wt) Aβ42. In addition, the A2V substitution caused Aβ40 to oligomerize similar to that of wt Aβ42, as evidenced by the formation of dimers, tetramers, hexamers, and dodecamers. In contrast, wt Aβ40 formed only dimers and tetramers. These results provide a basis for understanding how these two mutations lead to, or protect against, AD. They also suggest that the Aβ N-terminus, in addition to the oft discussed central hydrophobic cluster and C-terminus, can play a key role in controlling disease susceptibility

Amyloid β‑Protein Assembly: The Effect of Molecular Tweezers CLR01 and CLR03

The early oligomerization of amyloid β-protein (Aβ) has been shown to be an important event in the pathology of Alzheimer’s disease (AD). Designing small molecule inhibitors targeting Aβ oligomerization is one attractive and promising strategy for AD treatment. Here we used ion mobility spectrometry coupled to mass spectrometry (IMS-MS) to study the different effects of the molecular tweezers CLR01 and CLR03 on Aβ self-assembly. CLR01 was found to bind to Aβ directly and disrupt its early oligomerization. Moreover, CLR01 remodeled the early oligomerization of Aβ42 by compacting the structures of dimers and tetramers and as a consequence eliminated higher-order oligomers. Unexpectedly, the negative-control derivative, CLR03, which lacks the hydrophobic arms of the tweezer structure, was found to facilitate early Aβ oligomerization. Our study provides an example of IMS as a powerful tool to study and better understand the interaction between small molecule modulators and Aβ oligomerization, which is not attainable by other methods, and provides important insights into therapeutic development of molecular tweezers for AD treatment

Online Ozonolysis Combined with Ion Mobility-Mass Spectrometry Provides a New Platform for Lipid Isomer Analyses

One of the most significant challenges in contemporary lipidomics lies in the separation and identification of lipid isomers that differ only in site(s) of unsaturation or geometric configuration of the carbon–carbon double bonds. While analytical separation techniques including ion mobility spectrometry (IMS) and liquid chromatography (LC) can separate isomeric lipids under appropriate conditions, conventional tandem mass spectrometry cannot provide unequivocal identification. To address this challenge, we have implemented ozone-induced dissociation (OzID) in-line with LC, IMS, and high resolution mass spectrometry. Modification of an IMS-capable quadrupole time-of-flight mass spectrometer was undertaken to allow the introduction of ozone into the high-pressure trapping ion funnel region preceding the IMS cell. This enabled the novel LC-OzID-IMS-MS configuration where ozonolysis of ionized lipids occurred rapidly (10 ms) without prior mass-selection. LC-elution time alignment combined with accurate mass and arrival time extraction of ozonolysis products facilitated correlation of precursor and product ions without mass-selection (and associated reductions in duty cycle). Unsaturated lipids across 11 classes were examined using this workflow in both positive and negative ion modalities, and in all cases, the positions of carbon–carbon double bonds were unequivocally assigned based on predictable OzID transitions. Under these conditions, geometric isomers exhibited different IMS arrival time distributions and distinct OzID product ion ratios providing a means for discrimination of <i>cis/trans</i> double bonds in complex lipids. The combination of OzID with multidimensional separations shows significant promise for facile profiling of unsaturation patterns within complex lipidomes including human plasma

La Petite presse : journal quotidien... / [rédacteur en chef : Balathier Bragelonne]

07 juillet 18771877/07/07 (A11,N4078)

Additional file 2: Table S1. of Combined strategies for improving expression of Citrobacter amalonaticus phytase in Pichia pastoris

Primers, vectors and strains used in this study. (PDF 254 kb

Mass spectrometry profiling of pentosan polysulfate sodium (PPS) (ASMS 2017)

Pentosan polysulfate (PPS) is a semisynthetic heterogenous sulfated polysaccharide derived from xylan, the β-1,4-linked polymer of xylose. PPS sold by the brand name Elmiron in United States is taken orally to alleviate pain associated with interstitial cystitis. PPS is a mixture of hundreds or more discrete molecules built from a range of oligoxylose lengths modified with different combinations of functional group modifications, including sulfation, 4-O-methyl-glucuronidylation, acetylation, and others. The overall goal of our research is to develop an approach using MS together with other methods such as NMR to profile PPS at the molecular level. Profiling PPS according to its molecular composition would be invaluable for understanding biological activity, bioavailability, and pharmacokinetics, as well as for quality control.One Elmiron (100 mg PPS) capsule was extracted with 1 ml of HPLC-grade water, and further dilutions were made with this stock solution. Diluted PPS at a concentration of 0.5mg/ml was treated with an ion exchange resin for few hours, centrifuged and the supernatant collected. To this supernatant butylamine (15mM) and hexafluoroisopropanol (60mM) were added as an ion-pair reagent (final pH ~8.5). The treated sample was fractionated on C18 SPE cartridge using acetonitrile (ACN) starting from concentration of 10% up to 100% ACN. Each fraction was individually analyzed by FTICR and IMS-MS both in positive and negative mode. Agilent drift tube-IMS-QTOF MS and home-built drift tube IMS-MS were used to characterize PPS from different lots and locations of production.The mass spectrum obtained from PPS directly dissolved in water is complex and difficult to interpret due in-source fragmentation of sulfated oligosaccharides and presence of multiple metal ion adducts [M+Na]. We have explored the potential of ion-pair reversed phase chromatography to extract and analyze PPS using C18-SPE followed by MS detection using FTICR and IMS. When each eluate was injected directly in FTICR without any chromatographic separation, most of the PPS eluted in fraction containing 10% and 20% ACN. Analysis of mass spectra revealed presence of multiply charged state species, mostly +2, +3 and +4 for data collected in positive mode. Analysis of deconvulated peaks in positive mode displayed abundant neutral loss of 171.03 across the entire MS1 spectrum. This neutral loss of 171.03 units is most likely coming from the group –OSO₃NH₂(CH₂)₃CH₃ from PPS backbone. IMS-MS is capable of separating molecules that have the same mass-to-charge (m/z) ratio but different sizes, shapes or conformations. Therefore it is appealing for separating PPS with different polymerized sizes and different charge states and for reducing the complexity of mass spectra. Low-molecular-weight heparin, another sulfated oligosaccharide, was used as a standard to develop IMS-MS method. Heparin DP10 which has molecular weight around 3000 Da has shown a 2D IMS-MS spectrum with trend lines for charge +2 and +3 and m/z range from 1000 to 2000. Preliminary data of PPS showed 2D IMS-MS profiles with charge states from +1 to +5 and m/z range from 300 to 2500. These results show that IMS-MS can reduce the complexity of sulfated polysaccharide spectra by additional separation of different charge states and polysaccharide sizes. However the spectra are still complex for peak assignment without any pre-treatment. The uses of ion exchange resin and ion-pairs have shown improved sensitivity and separation in IMS-MS.<p></p

Amyloid β‑Protein Assembly and Alzheimer’s Disease: Dodecamers of Aβ42, but Not of Aβ40, Seed Fibril Formation

Evidence suggests that oligomers of the 42-residue form of the amyloid β-protein (Aβ), Aβ42, play a critical role in the etiology of Alzheimer’s disease (AD). Here we use high resolution atomic force microscopy to directly image populations of small oligomers of Aβ42 that occur at the earliest stages of aggregation. We observe features that can be attributed to a monomer and to relatively small oligomers, including dimers, hexamers, and dodecamers. We discovered that Aβ42 hexamers and dodecamers quickly become the dominant oligomers after peptide solubilization, even at low (1 μM) concentrations and short (5 min) incubation times. Soon after (≥10 min), dodecamers are observed to seed the formation of extended, linear preprotofibrillar β-sheet structures. The preprotofibrils are a single Aβ42 layer in height and can extend several hundred nanometers in length. To our knowledge this is the first report of structures of this type. In each instance the preprotofibril is associated off center with a single layer of a dodecamer. Protofibril formation continues at longer times, but is accompanied by the formation of large, globular aggregates. Aβ40, by contrast, does not significantly form the hexamer or dodecamer but instead produces a mixture of smaller oligomers. These species lead to the formation of a branched chain-like network rather than discrete structures

Role of Species-Specific Primary Structure Differences in Aβ42 Assembly and Neurotoxicity

A variety of species express the amyloid β-protein (Aβ (the term “Aβ” refers both to Aβ40 and Aβ42, whereas “Aβ40” and “Aβ42” refer to each isoform specifically). Those species expressing Aβ with primary structure identical to that expressed in humans have been found to develop amyloid deposits and Alzheimer’s disease-like neuropathology. In contrast, the Aβ sequence in mice and rats contains three amino acid substitutions, Arg5Gly, His13Arg, and Tyr10Phe, which apparently prevent the development of AD-like neuropathology. Interestingly, the brush-tailed rat, Octodon degus, expresses Aβ containing only one of these substitutions, His13Arg, and <i>does</i> develop AD-like pathology. We investigate here the biophysical and biological properties of Aβ peptides from humans, mice (Mus musculus), and rats (Octodon degus). We find that each peptide displays statistical coil → β-sheet secondary structure transitions, transitory formation of hydrophobic surfaces, oligomerization, formation of annuli, protofibrils, and fibrils, and an inverse correlation between rate of aggregation and aggregate size (faster aggregation produced smaller aggregates). The rank order of assembly rate was mouse > rat > Aβ42. The rank order of neurotoxicity of assemblies formed by each peptide immediately after preparation was Aβ42 > mouse ≈ rat. These data do <i>not</i> support long-standing hypotheses that the primary factor controlling development of AD-like neuropathology in rodents is Aβ sequence. Instead, the data support a hypothesis that assembly quaternary structure <i>and</i> organismal responses to toxic peptide assemblies mediate neuropathogenetic effects. The implication of this hypothesis is that a valid understanding of disease causation within a given system (organism, tissue, etc.) requires the coevaluation of both biophysical and cell biological properties of that system

Fundamentals of Ion Dynamics in Structures for Lossless Ion Manipulations (ASMS 2016)

<p>While much effort has gone into developing improved separation strategies for use with MS analysis, the extensive demands for more effective characterization of complex biological mixtures drives further efforts to meet these needs. Gas phase separations based upon ion mobility (IM) are fast, amenable to high-throughput application, and provide high reproducibility. New platforms that allow complex ion manipulations, e.g. mobility based ion selections, CID, ion/ion reactions, in addition to higher resolution separations, are of interest. Here we characterize the fundamentals of ion dynamics and consider novel ion processing approaches in Structures for Lossless Ion Manipulations (SLIM). Ion confinement, ion dynamics, heating effects and separation performance and other insights from simulations and theory will be discussed.</p

Collision Cross Section Calibration with Structures for Lossless Ion Manipulations (ASMS 2017)

Ion mobility mass spectrometry (IM-MS) is a powerful separation and structural characterization technique, providing the ability to measure collision cross sections (CCS), revealing information about the three dimensional structure of gaseous ions. In many cases, CCS can be used to identify ions in a mixture, and highly accurate and precise CCS measurements greatly expand IM-MS capabilities. Recently, long path structures for lossless ion manipulations (SLIM) traveling wave (TW) IM modules have allowed extremely high resolution IM separations. However, since SLIM do not utilize uniform low-field drift cells, CCS cannot be directly measured from experiments. To that end, we have developed a CCS calibration framework to provide high resolution CCS assignment.Travelling wave potentials and a combination of lateral DC-only electrodes (guards) and extended RF electrodes aligned with the ion path provided for TWIM separations in several Torr nitrogen in conjunction with efficient ion confinement. Ions from nanoelectrospray ionization of mixtures of multiple classes of compounds (e.g. peptide, glycan, lipid) were injected to the SLIM module. A SLIM ion switch controlled whether ions made multiple passes through the serpentine path of the module, or were sent to the TOF MS for analysis. Multiple mixtures of calibrants of different classes overlapping in CCS space with the compounds studied were prepared and infused as both external and internal calibrants. TWIM-MS features were extracted and calibrated using in-house developed software tools.Recently, multi-pass SLIM separations have been reported, showing very high IM resolutions and peak capacities for a variety of compounds, including peptides, lipids, and carbohydrates. A SLIM ion switch was positioned at the end of a long (>10 meter) serpentine ion path to allow ions to either exit to a TOF MS for mass analysis, or to be shuttled to the beginning of the ion path for addition separation. Resolutions much higher than that from conventional commercially available instruments (both TW and uniform field) have been achieved (e.g., separation powers of over 1000 for singly charged ions for 200 m multi-pass separations). Due to the abundance of information from bottom-up proteomics of many protein standards (e.g. tryptic peptide accurate monoisotopic MW), the first efforts for applying CCS calibration have utilized whole protein digests. Early results have shown baseline separations of peptides in a protein digest (serum albumin) that are inseparable by conventional IM instruments. Initially, a poly-alanine mixture was used to begin evaluating CCS calibrations for peptides and was used as external and internal calibration standards. The protein digest was then run on an Agilent 6560 IM-MS to compare the calibrated CCS values against values measured directly by a uniform low field instrument. The presentation will detail the efficacy of CCS calibration in SLIM TWIM measurements as well as effects resulting from the choice of calibrant, internal vs. external calibration, and other biological compound classes