Analytical Challenge 3

Analytical Challenge 3     

Direct Nanospectroscopic Verification of the Amyloid Aggregation Pathway

The aggregation pathways of neurodegenerative peptides determine the disease etiology, and their better understanding can lead to strategies for early disease treatment. Previous research has allowed modelling of hypothetic aggregation pathways. However, their direct experimental observation has been elusive owing to methodological limitations. Herein, we demonstrate that nanoscale chemical mapping by tip‐enhanced Raman spectroscopy of single amyloid fibrils at various stages of aggregation captures the fibril formation process. We identify changes in TERS/Raman marker bands for Aβ_1‐42, including the amide III band (above 1255 cm^?1 for turns/random coil and below 1255 cm^?1 for β‐sheet conformation). The spatial distribution of β‐sheets in aggregates is determined, allowing verification of a particular fibrillogenesis pathway, starting from aggregation of monomers to meta‐stable oligomers, which then rearrange to ordered β‐sheets, already at the oligomeric or protofibrillar stage.     

A Radical-Mediated Pathway for the Formation of [M + H]<Superscript>+</Superscript> in Dielectric Barrier Discharge Ionization

Active capillary plasma ionization is a highly efficient ambient ionization method. Its general principle of ion formation is closely related to atmospheric pressure chemical ionization (APCI). The method is based on dielectric barrier discharge ionization (DBDI), and can be constructed in the form of a direct flow-through interface to a mass spectrometer. Protonated species ([M + H]⁺) are predominantly formed, although in some cases radical cations are also observed. We investigated the underlying ionization mechanisms and reaction pathways for the formation of protonated analyte ([M + H]⁺). We found that ionization occurs in the presence and in the absence of water vapor. Therefore, the mechanism cannot exclusively rely on hydronium clusters, as generally accepted for APCI. Based on isotope labeling experiments, protons were shown to originate from various solvents (other than water) and, to a minor extent, from gaseous impurities and/or self-protonation. By using CO₂ instead of air or N₂ as plasma gas, additional species like [M + OH]⁺ and [M ? H]⁺ were observed. These gas-phase reaction products of CO₂ with the analyte (tertiary amines) indicate the presence of a radical-mediated ionization pathway, which proceeds by direct reaction of the ionized plasma gas with the analyte. The proposed reaction pathway is supported with density functional theory (DFT) calculations. These findings add a new ionization pathway leading to the protonated species to those currently known for APCI.

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Discriminating alkylbenzene isomers with tandem mass spectrometry using a dielectric barrier discharge ionization source

Soft ambient ionization sources generate reactive species that interact with analyte molecules to form intact molecular ions, which allows rapid, sensitive, and direct identification of the molecular mass. We used a dielectric barrier discharge ionization (DBDI) source with nitrogen at atmospheric pressure to detect alkylated aromatic hydrocarbon isomers (C₈H₁₀ or C₉H₁₂). Intact molecular ions [M]^•+ were detected at 2.4 kV_pp, but at increased voltage (3.4 kV_pp), [M + N]⁺ ions were formed, which could be used to differentiate regioisomers by collision-induced dissociation (CID). At 2.4 kV_pp, alkylbenzene isomers with different alkyl-substituents could be identified by additional product ions: ethylbenzene and -toluene formed [M-2H]⁺, isopropylbenzene formed abundant [M-H]⁺, and propylbenzene formed abundant C₇H₇⁺. At an operating voltage of 3.4 kV_pp, fragmentation of [M + N]⁺ by CID led to neutral loss of HCN and CH₃CN, which corresponded to steric hindrance for excited state N-atoms approaching the aromatic ring (C-H). The ratio of HCN to CH₃N loss (interday relative standard deviation [RSD] < 20%) was distinct for ethylbenzene and ethyltoluene isomers. The greater the number of alkyl-substituents (C-CH₃) and the more sterically hindered (meta > para > ortho) the aromatic core, the greater the loss of CH₃CN relative to HCN was.     

Rapid classification of perfumes by extractive electrospray ionization mass spectrometry (EESI-MS)

Extractive electrospray ionization mass spectrometry (EESI-MS) was applied to rapid fingerprinting of various perfumes for quality classification. Unique EESI-MS fingerprints of ten famous brands were obtained. This technique was shown to be applicable to rapid forgery detection on the example of an authentic and a counterfeit 'Miss Dior' fragrance by Christian Dior. We believe that the high throughput and simplicity of this sample-preparation-free method can be advantageous in the perfume industry, for instance when applied to online quality control.     

Critical evaluation of mass spectrometric measurement of dissociation constants: accuracy and cross-validation against surface plasmon resonance and circular dichroism for the calmodulin-melittin system

We present a comprehensive study for determining the binding affinity of a protein-ligand complex, using mass spectrometric methods. Mass spectrometry has been used to study noncovalent interactions for a number of years. However, the use of soft ionization mass spectrometry for quantitative analysis of noncovalently bound complexes is not widely accepted. This paper reports a comparison of MS methods against established methods such as surface plasmon resonance (SPR) and circular dichroism (CD) whose suitability for the quantitative assessment of noncovalent interactions is well known. ESI titration and MALDI-SUPREX were used as representative mass spectrometric methods for this work. We chose to study the calmodulin-melittin complex that presents three challenges: (i) it exhibits a high affinity (low nanomolar KD); (ii) complexes are formed only in the presence of a coactivator, calcium ions in this case; and (iii) the protein and the complex show a different ionization efficiency. Dissociation constants were obtained from each method for the selected system and compared thoroughly to elucidate pros and cons of the selected methodologies in terms of their ability for the determination of binding constants of protein-ligand complexes. ESI titration, SPR, CD and MALDI-SUPREX yielded KD values in the low nanomolar range that are in general agreement with an older value reported in the literature. We also critically evaluated the limitations in particular of the MS methods and the associated data evaluation procedures. We present an improved evaluation of SUPREX data, as well as a detailed error analysis for all methods used.     

Exploring fluorescence and fragmentation of ions produced by electrospray ionization in ultrahigh vacuum

An instrument for fluorescence spectrometry and FTICR-MS has been developed to study ions produced by a commercial ESI source.

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Hexameric supramolecular scaffold orients carbohydrates to sense bacteria

Carbohydrates are integral to biological signaling networks and cell-cell interactions, yet the detection of discrete carbohydrate-lectin interactions remains difficult since binding is generally weak. A strategy to overcome this problem is to create multivalent sensors, where the avidity rather than the affinity of the interaction is important. Here we describe the development of a series of multivalent sensors that self-assemble via hydrophobic supramolecular interactions. The multivalent sensors are comprised of a fluorescent ruthenium(II) core surrounded by a heptamannosylated β-cyclodextrin scaffold. Two additional series of complexes were synthesized as proof-of-principle for supramolecular self-assembly, the fluorescent core alone and the core plus β-cyclodextrin. Spectroscopic analyses confirmed that the three mannosylated sensors displayed 14, 28, and 42 sugar units, respectively. Each complex adopted original and unique spatial arrangements. The sensors were used to investigate the influence of carbohydrate spatial arrangement and clustering on the mechanistic and qualitative properties of lectin binding. Simple visualization of binding between a fluorescent, multivalent mannose complex and the Escherichia coli strain ORN178 that possesses mannose-specific receptor sites illustrates the potential for these complexes as biosensors.     

Compelling Advantages of Negative Ion Mode Detection in High-Mass MALDI-MS for Homomeric Protein Complexes

Chemical cross-linking in combination with high-mass MALDI mass spectrometry allows for the rapid identification of interactions and determination of the complex stoichiometry of noncovalent protein–protein interactions. As the molecular weight of these complexes increases, the fraction of multiply charged species typically increases. In the case of homomeric complexes, signals from multiply charged multimers overlap with singly charged subunits. Remarkably, spectra recorded in negative ion mode show lower abundances of multiply charged species, lower background, higher reproducibility, and, thus, overall cleaner spectra compared with positive ion mode spectra. In this work, a dedicated high-mass detector was applied for measuring high-mass proteins (up to 200 kDa) by negative ion mode MALDI-MS. The influences of sample preparation and instrumental parameters were carefully investigated. Relative signal integrals of multiply charged anions were relatively independent of any of the examined parameters and could thus be approximated easily for the spectra of cross-linked complexes. For example, the fraction of doubly charged anions signals overlapping with the signals of singly charged subunits could be more precisely estimated than in positive ion mode. Sinapinic acid was found to be an excellent matrix for the analysis of proteins and cross-linked protein complexes in both ion modes. Our results suggest that negative ion mode data of chemically cross-linked protein complexes are complementary to positive ion mode data and can in some cases represent the solution phase situation better than positive ion mode.     

High-density micro-arrays for mass spectrometry

Functional high-density micro-arrays for mass spectrometry enable rapid picolitre-volume aliquoting and ultrasensitive analysis of microscale samples, for example, single cells.