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While it is generally known that metabolic disorders and circadian dysfunction are intertwined, how the two systems affect each other is not well understood, nor are the genetic factors that might exacerbate this pathological interaction. Blood chemistry is profoundly changed in metabolic disorders, and we have previously shown that serum factors change cellular clock properties. To investigate if circulating factors altered in metabolic disorders have circadian modifying effects, and whether these effects are of genetic origin, we measured circadian rhythms in U2OS cell in the presence of serum collected from diabetic, obese or control subjects. We observed that circadian period lengthening in U2OS cells was associated with serum chemistry that is characteristic of insulin resistance. Characterizing the genetic variants that altered circadian period length by genome‐wide association analysis, we found that one of the top variants mapped to the E3 ubiquitin ligase MARCH1 involved in insulin sensitivity. Confirming our data, the serum circadian modifying variants were also enriched in type 2 diabetes and chronotype variants identified in the UK Biobank cohort. Finally, to identify serum factors that might be involved in period lengthening, we performed detailed metabolomics and found that the circadian modifying variants are particularly associated with branched chain amino acids, whose levels are known to correlate with diabetes and insulin resistance. Overall, our multi‐omics data showed comprehensively that systemic factors serve as a path through which metabolic disorders influence circadian system, and these can be examined in human populations directly by simple cellular assays in common cultured cells. [ABSTRACT FROM AUTHOR]

Rationale: Secondary-electrospray ionization (SESI) coupled with high-resolution mass spectrometry is a powerful tool for the discovery of biomarkers in exhaled breath. A primary electrospray consisting of aqueous formic acid (FA) is currently used to charge the volatile organic compounds in breath. To investigate whether alternate electrospray compositions could enable different metabolite coverage and sensitivities, the electrospray dopants NaI and AgNO3 were tested. Methods: In a proof-of-principle manner, the exhaled breath of one subject was analyzed repeatedly with different electrospray solutions and with the help of a spectral stitching technique. Capillary diameter and position were optimized to achieve proper detection of exhaled breath. The detected features were then compared using formula annotation. Using an evaporation-based gas standard system, the signal response of the different solutions was probed. Results: Principal component analysis revealed a substantial difference in features detected with AgNO3. With silver, more sulfur-containing features and more unsaturated hydrocarbon compounds were detected. Furthermore, more primary amines were potentially ionized, as indicated by van Krewelen diagrams. In total, twice as many features were unique to AgNO3 than for other electrospray dopants. Using gas standards at known concentrations, the high sensitivity of FA as a dopant was demonstrated but also indicated alternate sensitivities of the other electrospray solutions. Conclusions: This work demonstrated the potential of AgNO3 as a complementary dopant for further biomarker discovery in SESI-based breath analysis. [ABSTRACT FROM AUTHOR]

We doped nitrogen into monolayer graphene using reactive nitrogen species from a dielectric barrier discharge (DBD). After 30 s of treatment, the graphene monolayer had a moderate degree of damage (ID/IG = 1.2) and an increase in the N‐atom (pyrrolic) and O‐atom (mixed) content. During long treatment times (20 min), the treated area increased radially and the graphene was destroyed. Overall, the N‐atom content increased with increasing operating voltage of the DBD source. When the graphene was treated with only neutral reactive nitrogen species, the N‐atom content and type remained unchanged. Therefore, we hypothesize that the primary reactive species resulting in pyrrolic N‐doping from the DBD are neutrals such as N(4S) and possibly N(2P). [ABSTRACT FROM AUTHOR]

To analyze data acquired from experiments based on multifactorial designs, approaches able to decompose and model the main sources of variation in the measurements are particularly useful. Although methods combining analysis of variance (ANOVA) with principal components analysis (PCA) or simultaneous components analysis (SCA), namely, ANOVA‐PCA (APCA) and ANOVA‐SCA (ASCA), have proven to be efficient to tackle such data structures, they require the construction and interpretation of a model for each experimental factor and interaction. Other methods like parallel factor analysis‐SCA (PARAFASCA), ANOVA multiblock orthogonal partial least squares (AMOPLS) and ANOVA‐Common Dimensions (AComDim) offer the possibility to have an overall picture of all the sources of variation by means of a single model. Moreover, acquiring data from one or multiple data sources through the same experimental design is becoming more common, and approaches able to cope with such complex data structures are needed. In this tutorial, an explanation of the theory and software implementation of AComDim as well as its usage for the analysis of data acquired from experimental designs is provided. Relying on the multiblock nature of AComDim, an extension to cases where data from multiple sources are acquired on the same samples is proposed. [ABSTRACT FROM AUTHOR]

A novel chemometric approach is proposed to analyze high‐dimensional data from unbalanced designs of experiments. It combines a rebalancing strategy based on averages with the ASCA method under the name Rebalanced ASCA (RASCA). The ability of RASCA to handle unbalanced designs was compared with standard ASCA, as well as state‐of‐the‐art methods, such as ASCA+ and WE‐ASCA. For that purpose, a controlled framework was designed to provide a systematic comparison of the various approaches. It included two real datasets obtained from initially balanced designs, which were gradually unbalanced by removing observations belonging to specific combinations of factor levels. The results illustrate that all methods considered led to identical solutions when the initial balanced design was kept. Nevertheless, increasing differences appeared when the design was gradually unbalanced. The proposed benchmark showed that RASCA and ASCA+ provided overall similar results for all effects with high agreement with the balanced solutions in comparison to classical ASCA and WE‐ASCA. RASCA was found to be a suitable chemometric tool to tackle unbalanced designs by ensuring unbiased parameter estimators with the added benefit of producing rigorously orthogonal effect matrices, thus facilitating interpretation. [ABSTRACT FROM AUTHOR]

Reactive oxygen species (ROS) are critical for many cellular functions, and dysregulation of ROS involves the development of multiple types of tumors, including pancreatic cancer. However, ROS have been grouped into a single biochemical entity for a long time, and the specific roles of certain types of ROS in tumor cells (e.g. pancreatic ductal adenocarcinoma (PDAC)) have not been systematically investigated. In this work, a highly sensitive and accurate mass spectrometry‐based method was applied to study PDAC cells of humans and of genetically modified animals. The results show that the oncogenic KRAS mutation promotes the accumulation of hydrogen peroxide (H2O2) rather than superoxide or hydroxyl radicals in pancreatic cancer cells. We further identified that the enriched H2O2 modifies cellular metabolites and promotes the survival of pancreatic cancer cells. These findings highlight the specific roles of H2O2 in pancreatic cancer development, which may provide new directions for pancreatic cancer therapy. [ABSTRACT FROM AUTHOR]

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 kVpp, but at increased voltage (3.4 kVpp), [M + N]+ ions were formed, which could be used to differentiate regioisomers by collision‐induced dissociation (CID). At 2.4 kVpp, 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 kVpp, 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. [ABSTRACT FROM AUTHOR]

Lack of appropriate tools for visualizing cell membrane molecules at the nanoscale in a non‐invasive and label‐free fashion limits our understanding of many vital cellular processes. Here, we use tip‐enhanced Raman spectroscopy (TERS) to visualize the molecular distribution in pancreatic cancer cell (BxPC‐3) membranes in ambient conditions without labelling, with a spatial resolution down to ca. 2.5 nm. TERS imaging reveals segregation of phenylalanine‐, histidine‐, phosphatidylcholine‐, protein‐, and cholesterol‐rich BxPC‐3 cell membrane domains at the nm length‐scale. TERS imaging also showed a cell membrane region where cholesterol is mixed with protein. Interestingly, the higher resolution TERS imaging revealed that the molecular domains observed on the BxPC‐3 cell membrane are not chemically "pure" but also contain other biomolecules. These results demonstrate the potential of TERS for non‐destructive and label‐free imaging of cell membranes with nanoscale resolution. [ABSTRACT FROM AUTHOR]

Rationale: The co‐fragmentation of precursors in direct infusion (DI) tandem high‐resolution mass spectrometry (HRMS) can complicate the fragment spectra and consequently lead to false hits during compound identification. Methods: The method herein described, termed IQAROS (incremental quadrupole acquisition to resolve overlapping spectra), modulates the intensities of precursors and fragments by stepwise movement of the quadrupole isolation window over the mass‐to‐charge (m/z) range of the precursors. The modulated signals are then deconvoluted by a linear regression model to reconstruct the fragment spectra with less interference. The hardware to demonstrate the use of IQAROS was an orbitrap with electrospray ionization (ESI) or secondary electrospray ionization (SESI), although the method can also be applied to other ionization techniques or mass analyzers. Results: Assessing the performance of IQAROS with isobaric standards revealed that the reconstructed fragment spectra match with spectra acquired from the pure standards and that more compounds were correctly identified compared with the classical approach with the quadrupole centered at the m/z value of the precursor of interest. Moreover, the strength of IQAROS is exemplified by the identification of two isobaric biomarkers directly from a breath sample with SESI‐HRMS. Conclusions: With IQAROS, cleaner fragment spectra of co‐fragmenting isobars during DI‐HRMS analysis can be obtained. IQAROS can easily be set up by the standard graphical user interface of the instrument. Therefore, it facilitates the characterization of features of interest in samples analyzed by DI‐HRMS, for example, in high‐throughput or real‐time metabolomics. [ABSTRACT FROM AUTHOR]

Native electrospray ionization (ESI) and nanoelectrospray ionization (nESI) allow researchers to analyze intact biomolecules and their complexes by mass spectrometry (MS). The data acquired using these soft ionization techniques provide a snapshot of a given biomolecules structure in solution. Over the last thirty years, several nESI and ESI sources capable of controlling spray solution temperature have been developed. These sources can be used to elucidate the thermodynamics of a given analyte, as well as provide structural information that cannot be readily obtained by other, more commonly used techniques. This review highlights how the field of temperature‐controlled mass spectrometry has developed. [ABSTRACT FROM AUTHOR]

Transition metal carbides and nitrides (MXenes) have shown great potential for constructing thin, high‐performance electromagnetic interference (EMI) shields. The challenges with these materials involve the weak interfacial interactions of MXenes, which results in inferior mechanical properties and structure of the MXene films and a conductivity/EMI shielding performance decay related to the poor MXene oxidation stability. Numerous efforts have been devoted to improving the mechanical properties or oxidation stability of the films, which always comes at the expense of EMI shielding performance. Here, ultrafine (≈1.4 nm) cellulose nanofibers are employed to achieve physical and chemical dual cross‐linking of MXene (PC‐MXene) nanosheets. The procedure involves drying of flexible and highly conductive PC‐MXene films at ambient pressure and is energy‐efficient and scalable. Compared to the MXene films, the PC‐MXene films show significantly improved mechanical strength, hydrophobicity, oxidation stability, and are waterproof, without compromising the excellent EMI shielding effectiveness (SE). Moreover, the freestanding PC‐MXene films reach a thickness of merely 0.9 µm and exhibit a high SE of 33.3 dB, which cannot be achieved by pure MXene films. This leads to ultrahigh thickness‐specific SE and surface‐specific SE values of 37 000 dB mm−1 and 148 000 dB cm2 g−1 respectively, significantly surpassing those of previously reported MXene‐based films. [ABSTRACT FROM AUTHOR]

Even populations of clonal cells are heterogeneous, which requires high‐throughput analysis methods with single‐cell sensitivity. Here, we propose a rapid, label‐free single‐cell analytical method based on active capillary dielectric barrier discharge ionization mass spectrometry, which can analyze multiple metabolites in single cells at a rate of 38 cells/minute. Multiple cell types (HEK‐293T, PANC‐1, CFPAC‐1, H6c7, HeLa and iBAs) were discriminated successfully. We found evidence for abnormal lipid metabolism in pancreatic cancer cells. We also analyzed gene expression in a cancer genome atlas dataset and found that the mRNA level of a critical enzyme of lipid synthesis (ATP citrate lyase, ACLY) was upregulated in human pancreatic ductal adenocarcinoma (PDAC). Moreover, both an ACLY chemical inhibitor and a siRNA approach targeting ACLY could suppress the viability of PDAC cells. A significant reduction in lipid content in treated cells indicates that ACLY could be a potential target for treating pancreatic cancer. [ABSTRACT FROM AUTHOR]

Visualizing the molecular organization of lipid membranes is essential to comprehend their biological functions. However, current analytical techniques fail to provide a non‐destructive and label‐free characterization of lipid films under ambient conditions at nanometer length scales. In this work, we demonstrate the capability of tip‐enhanced Raman spectroscopy (TERS) to probe the molecular organization of supported DPPC monolayers on Au (111), prepared using the Langmuir–Blodgett (LB) technique. High‐quality TERS spectra were obtained, that permitted a direct correlation of the topography of the lipid monolayer with its TERS image for the first time. Furthermore, hyperspectral TERS imaging revealed the presence of nanometer‐sized holes within a continuous DPPC monolayer structure. This shows that a homogeneously transferred LB monolayer is heterogeneous at the nanoscale. Finally, the high sensitivity and spatial resolution down to 20 nm of TERS imaging enabled reproducible, hyperspectral visualization of molecular disorder in the DPPC monolayers, demonstrating that TERS is a promising nanoanalytical tool to investigate the molecular organization of lipid membranes. [ABSTRACT FROM AUTHOR]

Quadruplexes are non‐canonical nucleic acid structures essential for many cellular processes. Hybrid quadruplex–duplex oligonucleotide assemblies comprised of multiple domains are challenging to study with conventional biophysical methods due to their structural complexity. Here, we introduce a novel method based on native mass spectrometry (MS) coupled with a custom‐built temperature‐controlled nanoelectrospray ionization (TCnESI) source designed to investigate interactions between proximal DNA domains. Thermal denaturation experiments were aimed to study unfolding of multi‐stranded oligonucleotide constructs derived from biologically relevant structures and to identify unfolding intermediates. Using the TCnESI MS, we observed changes in Tm and thermodynamic characteristics of proximal DNA domains depending on the number of domains, their position, and order in a single experiment. [ABSTRACT FROM AUTHOR]

Rationale: Fragrances are organic compounds with pleasant odors that are widely used in every aspect of our daily life; some fragrance ingredients can cause allergic reactions. Hence, the qualitative and quantitative analysis of fragrance allergens can prevent consumers coming into contact with these compounds. In this study, we evaluated the ability of a dielectric barrier discharge ionization (DBDI) source for analyzing allergens that occur in fragrances. Methods: A home‐built liquid‐infusion device was used to evaporate the liquid samples. An active capillary plasma ionization source, which is based on a dielectric barrier discharge, was used to ionize the analytes. Mass spectra were acquired in positive ion mode with an LTQ Orbitrap mass spectrometer. Results: Seven typical fragrance allergens were analyzed in this study. The limits of detections (LODs) were as low as 0.0001 ppm and a linear dynamic range of 2–3 orders of magnitude was achieved. Allergens in five different perfume products were successfully analyzed and quantified by this method, with analysis times of less than 1 min per sample. Conclusions: This work introduces a DBDI‐MS‐based analytical method for detecting and quantifying fragrance allergens. Since DBDI has the advantages of high sensitivity, simple operation and fast analysis time, it is very suitable for the rapid analysis of trace allergens in fragrances, and could easily be used for quality control of consumer products in the cosmetics market. [ABSTRACT FROM AUTHOR]

Abnormal aggregation of amyloid‐β is a very complex and heterogeneous process. Owing to methodological limitations, the aggregation pathway is still not fully understood. Herein a new approach is presented in which the secondary structure of single amyloid‐β aggregates is investigated with tip‐enhanced Raman spectroscopy (TERS) in a liquid environment. Clearly resolved TERS signatures of the amide I and amide III bands enabled a detailed analysis of the molecular structure of single aggregates at each phase of the primary aggregation of amyloid‐β and also of small species on the surface of fibrils attributed to secondary nucleation. Notably, a β‐sheet rearrangement from antiparallel in protofibrils to parallel in fibrils is observed. This study allows better understanding of Alzheimer's disease etiology and the methodology can be applied in studies of other neurodegenerative disorders. [ABSTRACT FROM AUTHOR]

Stabilizing the DNA and RNA structures known as G‐quadruplexes (G4s) using specific ligands is a strategy that has been proposed to fight cancer. However, although G‐quadruplex:ligand (G4:L) interactions have often been investigated, whether or not ligands are able to disrupt G‐quadruplex:protein (G4:P) interactions remains poorly studied. In this study, using native mass spectrometry, we have investigated ternary G4:L:P complexes formed by G4s, some of the highest affinity ligands, and the binding domain of the RHAU helicase. Our results suggest that RHAU binds not only preferentially to parallel G4s, but also to free external G‐quartets. We also found that, depending on the G4, ligands could prevent the binding of the peptide, either by direct competition for the binding sites (orthosteric inhibition) or by inducing conformational changes (allosteric inhibition). Notably, the ligand Cu–ttpy (ttpy=4′‐tolyl‐2,2′:6′,2′′‐terpyridine) induced a conformational change that increased the binding of the peptide. This study illustrates that it is important to not only characterize drug–target interactions, but also how the binding to other partners is affected. [ABSTRACT FROM AUTHOR]

A capillary atmospheric pressure photoionization (cAPPI) source was used to analyze polycyclic aromatic hydrocarbons (PAHs) in complex matrices like grilled meat extract and urban dust reference material, as well as screening for PAHs in aqueous samples such as tap and lake water. A high‐throughput workflow was developed that allowed rapid screening of unknown samples by direct solid‐phase microextraction (SPME) coupled with cAPPI‐MS, with confirmatory gas chromatography performed only for samples containing trace amounts of PAHs. Extraction times were as low as 15 s, with a total analysis time of 2 min per sample for screening. Limits of detections were in the low pg/ml range and in the subpg/ml range for the direct and chromatographic approach, respectively, with a linear dynamic range between two and three orders of magnitude, as determined for 15 model PAHs. This rapid approach represents an attractive way to screen samples containing nonpolar compounds using an ambient ionization source. [ABSTRACT FROM AUTHOR]

Rationale: Ferritin is an iron storage protein assembly, usually formed by a 24‐subunit protein shell and an iron core. The ferritin shell has been well studied using various structural biology tools such as X‐ray diffraction and cryo‐electron microscopy, whereas the iron status of ferritin is less studied and no well‐established method exists for characterizing the distribution of the iron loading of ferritin. Recent advances in mass spectrometry (MS) have expanded the observable m/z range, making the measurement of ferritin possible with MS. In this study, matrix‐assisted laser desorption ionization (MALDI)‐MS was employed to quantify the iron content of ferritin. Methods: The iron content of ferritin was quantified using a MALDI‐MS system coupled with a commercially available ion conversions dynode high‐mass detector. IgG1 antibody and its aggregates were used as external mass calibrants. The stability of HoloFt and ApoFt was also assessed in this study under different conditions, including various buffer pH, crosslinking agents and MALDI laser intensities. Results: The differences in peak width of HoloFt, ApoFt and IgG1 indicate the existence of mineral adducts in both HoloFt and ApoFt, and the mineral loading is heterogeneous among the HoloFt and ApoFt population. An average of 2773 ± 1584 iron atoms were determined for a commercial HoloFt sample. The iron core inside the ferritin complex is shown to stabilize and maintain the intact globular complex structure of ferritin. Conclusions: This work introduces a MALDI‐MS‐based workflow for characterizing the ferritin iron loading pattern, which is meaningful for clinical analysis of iron deficiency/overload. In addition, the stability of ferritin is examined under various conditions, providing a guideline for further method development related to ferritin complex. [ABSTRACT FROM AUTHOR]

Background and objective: Diagnosis of idiopathic pulmonary fibrosis (IPF) is complex and its pathogenesis is poorly understood. Recent findings indicate elevated levels of proline and other amino acids in lung tissue of IPF patients which may also be of diagnostic value. Following these findings, we hypothesized that such altered metabolic profiles would be mirrored in exhaled breath and could therefore be captured non‐invasively in real time. Methods: We aimed to validate these results using real‐time exhaled breath analysis by secondary electrospray ionization‐mass spectrometry, which can provide a non‐invasive, painless and fast insight into the metabolism. Breath analysis was performed in a matched 1:1 case–control study involving 21 patients with IPF and 21 control subjects. Results: We found significantly (P < 0.05) elevated levels of proline, 4‐hydroxyproline, alanine, valine, leucine/isoleucine and allysine in breath of IPF patients, whereas pyroglutamic acid and phenylalanine did not show significant differences. This coincides with the amino acid's abundance in pulmonary tissue indicating that our observations reflect progressing fibrosis. In addition, amino acid levels correlated across subjects, further supporting a common underlying pathway. We were able to obtain a cross‐validated area under the curve of 0.86, suggesting that these increased amino acid levels in exhaled breath have the potential to be used as biomarkers for IPF. Conclusion: We could validate previous findings of elevated lung tissue amino acid levels in IPF and show that online breath analysis might be a practical tool for a rapid screening for IPF. [ABSTRACT FROM AUTHOR]

A computational technique based on a simulated molecular evolution protocol was employed for anticancer peptide (ACP) design. Starting from known ACPs, innovative bioactive peptides were automatically generated in computer‐assisted design–synthesize–test cycles. This design algorithm offers a viable strategy for the generation of novel peptide sequences, without requiring a priori structure–activity knowledge. Sequence morphing and activity improvement were achieved through iterative amino acid variation and selection. Results show that not only the interaction of ACPs with the target membrane is important for their anticancer activity, but also the degree of peptide dimerization, which was corroborated by temperature profiling and electrospray mass spectrometry. Peptide design: Starting from known anticancer peptides (ACPs), new amino acid sequences were computationally generated and optimized in iterative design–synthesize–test cycles. The degree of peptide dimerization seems to play a critical role for membranolytic anticancer activity. [ABSTRACT FROM AUTHOR]

Resorcin[4]arene cavitands containing either 2,1,3‐benzotelluradiazole or 2,1,3‐benzothiadiazole motifs were dimerized to supramolecular capsules by chalcogen bonding. Their respective behavior varied depending on the interaction strength of the chalcogen bonds with Te forming strong interactions and S weak interactions. The tremendous strength of multiple 2Te–2N square interactions led to formation of a chalcogen‐bonded dimeric capsule in all solvents, as shown by X‐ray crystal structures with 16 short Te⋅⋅⋅N distances (≤2.9 Å) and confirmed by native electrospray ionization mass spectrometry (ESI‐MS). With the S cavitand, solvent‐dependent crystallization resulted in different arrangements: either a shifted 2S–2N square‐bonded capsule or an interlocked 1D polymer with an infinite π–π stacking array. The association constant to form the dimeric capsule in [D8]THF at 283 K, solely based on weak 2S–2N square interactions, was determined as Ka=786 m−1. Strengths and weaknesses: Dimeric chalcogen‐bonding capsules display a distinct behavior depending on the association strength, as shown by X‐ray structures and confirmed by NMR spectroscopy solution studies. While capsules based on weak S⋅⋅⋅N interactions rely on solvation and the associated hemisphere preorganization, capsular assemblies formed by strong Te⋅⋅⋅N interactions are beyond external influences. [ABSTRACT FROM AUTHOR]

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Abstract: 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 Aβ1‐42, 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. [ABSTRACT FROM AUTHOR]

Abstract: Tip‐enhanced Raman spectroscopy can provide molecular fingerprint information with ultrahigh spatial resolution, but the tip will be easily contaminated, thus leading to artifacts. It also remains a great challenge to establish tip‐enhanced fluorescence because of the quenching resulting from the proximity of the metal tip. Herein, we report shell‐isolated tip‐enhanced Raman and fluorescence spectroscopies by employing ultrathin shell‐isolated tips fabricated by atomic layer deposition. Such shell‐isolated tips not only show outstanding electromagnetic field enhancement in TERS but also exclude interference by contaminants, thus greatly promoting applications in solution. Tip‐enhanced fluorescence has also been achieved using these shell‐isolated tips, with enhancement factors of up to 1.7×103, consistent with theoretical simulations. Furthermore, tip‐enhanced Raman and fluorescence signals are acquired simultaneously, and their relative intensities can be manipulated by changing the shell thickness. This work opens a new avenue for ultrahigh resolution surface analysis using plasmon‐enhanced spectroscopies. [ABSTRACT FROM AUTHOR]

Abstract: An understanding of the photoisomerization mechanism of molecules bound to a metal surface at the molecular scale is required for designing photoswitches at surfaces. It has remained a challenge to correlate the surface structure and isomerization of photoswitches at ambient conditions. Herein, the photoisomerization of a self‐assembled monolayer of azobenzene‐thiol molecules on a Au surface was investigated using scanning tunneling microscopy and tip‐enhanced Raman spectroscopy. The unique signature of the cis isomer at 1525 cm−1 observed in tip‐enhanced Raman spectra was clearly distinct from the trans isomer. Furthermore, tip‐enhanced Raman images of azobenzene thiols after ultraviolet and blue light irradiation are shown with nanoscale spatial resolution, demonstrating a reversible conformational change. Interestingly, the cis isomers of azobenzene‐thiol molecules were preferentially observed at Au grain edges, which is confirmed by density functional theory. [ABSTRACT FROM AUTHOR]

This work describes a two-dimensional polymerization at an air/water interface and provides, for the first time, direct spectroscopic evidence for the kind of crosslinks formed and for the conversion reached in a covalently bonded monolayer sheet. This evidence was obtained through a combination of a variety of monolayer characterization techniques before and after transfer onto solid substrates, in particular by tip-enhanced Raman spectroscopy (TERS) and TERS mapping after transfer of both the monomer and polymer monolayer onto Au(111). This work is a major advance for the field of 2D polymers synthesized at the air/water interface as it, in principle, allows estimation of the crystallinity by percolation theory and the location of regions with defects. [ABSTRACT FROM AUTHOR]

The steady-state operation of Chinese hamster ovary (CHO) cells in perfusion bioreactors requires the equilibration of reactor dynamics and cell metabolism. Accordingly, in this work we investigate the transient cellular response to changes in its environment and their interactions with the bioreactor hydrodynamics. This is done in a benchtop perfusion bioreactor using MALDI-TOF MS through isotope labeling of complex intracellular nucleotides (ATP, UTP) and nucleotide sugars (UDP-Hex, UDP-HexNAc). By switching to a 13C6 glucose containing feed media during constant operation at 20 × 106 cells and a perfusion rate of 1 reactor volume per day, isotopic steady state was studied. A step change to the 13C6 glucose medium in spin tubes allowed the determination of characteristic times for the intracellular turnover of unlabeled metabolites pools [ABSTRACT FROM AUTHOR]

Chemical cross-linking combined with MALDI- MS was applied to structural analysis of a protein nanocontainer. Specifically, an engineered variant of lumazine synthase from Aquifex aeolicus (Aa LS-13) was investigated that self-assembles into a capsid-like structure and is known to encapsulate other proteins by Coulombic attraction. Two complementary soft ionization techniques, MALDI- MS and native ESI- MS, were utilized to map the subunit stoichiometry of the high molecular weight capsid. In accordance with the previously reported cryo-electron microscopy structure of this protein container, only pentameric subunits were detected. This study highlights the possibility to map subunit stoichiometry via chemical cross-linking with glutaraldehyde followed by MALDI- MS. The same approach was used to study protein-protein interactions during encapsulation of GFP(+36) by the Aa LS-13 capsid. Heterocomplexes between GFP(+36) and Aa LS-13 multimers were not observed when mixed at maximal loading capacity (AalS-13 monomer: GFP(+36) 4:1). This is in agreement with the known fast encapsulation of GFP(+36) by the protein capsid, which essentially removes any free GFP(+36) from the solution. Exceeding the maximal loading capacity by addition of excess GFP(+36) results in aggregation. [ABSTRACT FROM AUTHOR]

We report an investigation of interfacial fluorinated hydrocarbon (carboxylic-fantrip) monolayers by nanoscale imaging using tip-enhanced Raman spectroscopy (TERS) and density functional theory (DFT) calculations. By comparing TERS images of a sub-monolayer prepared by spin-coating and a π-π-stacked monolayer on Au(111) in which the molecular orientation is confined, specific Raman peaks shift and line widths narrow in the transferred LB monolayer. Based on DFT calculations that take into account dispersion corrections and surface selection rules, these specific effects are proposed to originate from π-π stacking and molecular orientation restriction. TERS shows the possibility to distinguish between a random and locked orientation with a spatial resolution of less than 10 nm. This work combines experimental TERS imaging with theoretical DFT calculations and opens up the possibility of studying molecular orientations and intermolecular interaction at the nanoscale and molecular level. [ABSTRACT FROM AUTHOR]

Perfusion cell culture processes allow the steady-state culture of mammalian cells at high viable cell density, which is beneficial for overall product yields and homogeneity of product quality in the manufacturing of therapeutic proteins. In this study, the extent of metabolic steady state and the change of the metabolite profile between different steady states of an industrial Chinese hamster ovary (CHO) cell line producing a monoclonal antibody (mAb) was investigated in stirred tank perfusion bioreactors. Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) of daily cell extracts revealed more than a hundred peaks, among which 76 metabolites were identified by tandem MS (MS/MS) and high resolution Fourier transform ion cyclotron resonance (FT-ICR) MS. Nucleotide ratios (Uridine (U)-ratio, nucleotide triphosphate (NTP)-ratio and energy charge (EC)) and multivariate analysis of all features indicated a consistent metabolite profile for a stable culture performed at 40 × 106 cells/mL over 26 days of culture. Conversely, the reactor was operated continuously so as to reach three distinct steady states one after the other at 20, 60, and 40 × 106 cells/mL. In each case, a stable metabolite profile was achieved after an initial transient phase of approximately three days at constant cell density when varying between these set points. Clear clustering according to cell density was observed by principal component analysis, indicating steady-state dependent metabolite profiles. In particular, varying levels of nucleotides, nucleotide sugar, and lipid precursors explained most of the variance between the different cell density set points. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:879-890, 2017 [ABSTRACT FROM AUTHOR]

Chemical analysis of aerosols collected from electronic cigarettes (ECs) has shown that these devices produce vapors that contain harmful and potentially harmful compounds. Conventional analytical methods used for the analysis of electronic cigarettes do not reflect the actual composition of the aerosols generated because they usually neglect the changes in the chemical composition that occur during the aerosol generation process and after collection. The aim of this work was to develop and apply a method for the real-time analysis of electronic cigarette aerosols, based on the secondary electrospray ionization technique coupled to high-resolution mass spectrometry, by mimicking the 'vaping' process. Electronic cigarette aerosols were successfully analyzed and quantitative differences were found between the liquids and aerosols. Thanks to the high sensitivity shown by this method, more than 250 chemical substances were detected in the aerosols, some of them showing a high correlation with the operating power of the electronic cigarettes. The method also allows proper quantification of several chemical components such as alkaloids and flavor compounds. [ABSTRACT FROM AUTHOR]

A two-dimensional covalent organic monolayer was synthesized from simple aromatic triamine and dialdehyde building blocks by dynamic imine chemistry at the air/water interface (Langmuir–Blodgett method). The obtained monolayer was characterized by optical microscopy, scanning electron microscopy, and atomic force microscopy, which unambiguously confirmed the formation of a large (millimeter range), unimolecularly thin aromatic polyimine sheet. The imine-linked chemical structure of the obtained monolayer was characterized by tip-enhanced Raman spectroscopy, and the peak assignment was supported by spectra simulated by density functional theory. Given the modular nature and broad substrate scope of imine formation, the work reported herein opens up many new possibilities for the synthesis of customizable 2D polymers and systematic studies of their structure- property relationships. [ABSTRACT FROM AUTHOR]

A two-dimensional covalent organic monolayer was synthesized from simple aromatic triamine and dialdehyde building blocks by dynamic imine chemistry at the air/water interface (Langmuir-Blodgett method). The obtained monolayer was characterized by optical microscopy, scanning electron microscopy, and atomic force microscopy, which unambiguously confirmed the formation of a large (millimeter range), unimolecularly thin aromatic polyimine sheet. The imine-linked chemical structure of the obtained monolayer was characterized by tip-enhanced Raman spectroscopy, and the peak assignment was supported by spectra simulated by density functional theory. Given the modular nature and broad substrate scope of imine formation, the work reported herein opens up many new possibilities for the synthesis of customizable 2D polymers and systematic studies of their structureproperty relationships. [ABSTRACT FROM AUTHOR]

Non-covalent interactions are essential for the structural organization of biomacromolecules and play an important role in molecular recognition processes, such as the interactions between proteins, glycans, lipids, DNA, and RNA. Mass spectrometry (MS) is a powerful tool for studying of noncovalent interactions, due to the low sample consumption, high sensitivity, and label-free nature. Nowadays, native-ESI MS is heavily used in studies of non-covalent interactions and to understand the architecture of biomolecular complexes. However, MALDI-MS is also becoming increasingly useful. It is challenging to detect the intact complex without fragmentation when analyzing non-covalent interactions with MALDI-MS. There are two methodological approaches to do so. In the first approach, different experimental and instrumental parameters are fine-tuned in order tofind conditions under which the complex is stable, such as applying non-acidic matrices and collecting first-shot spectra. In the second approach, the interacting species are "artificially" stabilized by chemical crosslinking. Both approaches are capable of studying noncovalently bound biomolecules even in quite challenging systems, such as membrane protein complexes. Herein, we review and compare native-ESI and MALDI MS for the study of non-covalent interactions. [ABSTRACT FROM AUTHOR]

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Noninvasive, real-time pharmacokinetic (PK) monitoring of ketamine, propofol, and valproic acid, and their metabolites was achieved in mice, using secondary electrospray ionization and high-resolution mass spectrometry. The PK profile of a drug influences its efficacy and toxicity because it determines exposure time and levels. The antidepressant and anaesthetic ketamine (Ket) and four Ket metabolites were studied in detail and their PK was simultaneously determined following application of different sub-anaesthetic doses of Ket. Bioavailability after oral administration vs. intraperitoneal injection was also investigated. In contrast to conventional studies that require many animals to be sacrificed even for low-resolution PK curves, this novel approach yields real-time PK curves with a hitherto unmatched time resolution (10 s), and none of the animals has to be sacrificed. This thus represents a major step forward not only in animal welfare, but also major cost and time savings. [ABSTRACT FROM AUTHOR]

RATIONALE: Up to now, there is no 'gold standard' for determining the resolution of a mass spectrometry imaging (MSI) setup (comprising the instrument, the sample preparation, the sample and the instrument settings). A standard sample in combination with a standard protocol to define the MSI resolution would be desirable in order to compare the setups of different laboratories, and as a regular quality control/performance check. METHODS: Microstructured resolution patterns were fabricated that can be used to determine the spatial resolution in MSI experiments, down to the range of a few μm. Two different strategies were employed, one where the resolution pattern is laser machined into a thin metal foil, which can be placed over a sample to be imaged, and a second one where hydrophilic grooves are machined into an omniphobic coating covering the surface of an indium tin oxide covered glass slide. When dragging a sample solution over the slide's surface, the sample is automatically retained in the hydrophilic grooves, but repelled by the omniphobic coating. RESULTS: The technology was tested on a commercial matrix-assisted laser desorption/ionization (MALDI) imaging instrument, and a spatial resolution in the vicinity of 50 μm was determined. The finest features of the microstructured resolution patterns are compatible with the best spatial resolution of MALDI imaging systems available to date. CONCLUSIONS: The use of metal resolution grids or glass slides with hydrophilic/hydrophobic structures is suitable for the convenient determination of the resolution limit of the MALDI imaging instrument as determined by its hardware. These structures are straightforward both to produce and to use. [ABSTRACT FROM AUTHOR]

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We present a novel and straightforward screening method to detect protein phosphorylations in complex protein mixtures. A proteolytic digest is separated by a conventional nanoscale liquid chromatography (nano-LC) separation and the eluate is immediately compartmentalized into microdroplets, which are spotted on a microarray MALDI plate. Subsequently, the enzyme alkaline phosphatase is applied to every second microarray spot to remove the phosphate groups from phosphorylated peptides, which results in a mass shift of n×−80 Da. The MALDI-MS scan of the microarray is then evaluated by a software algorithm to automatically identify the phosphorylated peptides by exploiting the characteristic chromatographic peak profile induced by the phosphatase treatment. This screening method does not require extensive MS/MS experiments or peak list evaluation and can be easily extended to other enzymatic or chemical reactions. [ABSTRACT FROM AUTHOR]

Future individualized patient treatment will need tools to monitor the dose and effects of administrated drugs. Mass spectrometry may become the method of choice to monitor drugs in real time by analyzing exhaled breath. This review describes the monitoring of exhaled drugs in real time by mass spectrometry. The biological background as well as the relevant physical properties of exhaled drugs are delineated. The feasibility of detecting and monitoring exhaled drugs is discussed in several examples. The mass spectrometric tools that are currently available to analyze breath in real time are reviewed. The technical needs and state of the art for on-site measurements by mass spectrometry are also discussed in detail. Off-line methods, which give support and are an important source of information for real-time measurements, are also discussed. Finally, some examples of drugs that have already been successfully detected in exhaled breath, including propofol, fentanyl, methadone, nicotine, and valproic acid are presented. Real-time monitoring of exhaled drugs by mass spectrometry is a relatively new field, which is still in the early stages of development. New technologies promise substantial benefit for future patient monitoring and treatment. © 2013 Wiley Periodicals, Inc. Mass Spec Rev 33: 394-413, 2014 [ABSTRACT FROM AUTHOR]

Chemical cross-linking, combined with mass spectrometry, has been applied to map three-dimensional protein structures and protein-protein interactions. Proper choice of the cross-linking agent, including its reactive groups and spacer arm length, is of great importance. However, studies to understand the details of reactivity of the chemical cross-linkers with proteins are quite sparse. In this study, we investigated chemical cross-linking from the aspects of the protein structures and the cross-linking reagents involved, by using two structurally well-known proteins, glyceraldehyde 3-phosohate dehydrogenase and ribonuclease S. Chemical cross-linking reactivity was compared using a series of homo- and hetero-bifunctional cross-linkers, including bis(sulfosuccinimidyl) suberate, dissuccinimidyl suberate, bis(succinimidyl) penta (ethylene glycol), bis(succinimidyl) nona (ethylene glycol), m-maleimidobenzoyl- N-hydroxysulfosuccinimide ester, 2-pyridyldithiol-tetraoxaoctatriacontane- N-hydrosuccinimide and succinimidyl-[( N-maleimidopropionamido)-tetracosaethyleneglycol]ester. The protein structure itself, especially the distances between target amino acid residues, was found to be a determining factor for the cross-linking efficiency. Moreover, the reactive groups of the chemical cross-linker also play an important role; a higher cross-linking reaction efficiency was found for maleimides compared to 2-pyrimidyldithiols. The reaction between maleimides and sulfhydryl groups is more favorable than that between N-hydroxysuccinimide esters and amine groups, although cysteine residues are less abundant in proteins compared to lysine residues. Copyright © 2013 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Copyright of Angewandte Chemie is the property of Wiley-Blackwell and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Methods for chemical analysis at the nanometer scale are crucial for understanding and characterizing nanostructures of modern materials and biological systems. Tip-enhanced Raman spectroscopy (TERS) combines the chemical information provided by Raman spectroscopy with the signal enhancement known from surface-enhanced Raman scattering (SERS) and the high spatial resolution of atomic force microscopy (AFM) or scanning tunneling microscopy (STM). A metallic or metallized tip is illuminated by a focused laser beam and the resulting strongly enhanced electromagnetic field at the tip apex acts as a highly confined light source for Raman spectroscopic measurements. This Review focuses on the prerequisites for the efficient coupling of light to the tip as well as the shortcomings and pitfalls that have to be considered for TERS imaging, a fascinating but still challenging way to look at the nanoworld. Finally, examples from recent publications have been selected to demonstrate the potential of this technique for chemical imaging with a spatial resolution of approximately 10 nm and sensitivity down to the single-molecule level for applications ranging from materials sciences to life sciences. [ABSTRACT FROM AUTHOR]

Most pesticides, herbicides and other plant treatment agents are applied to the crop surface. Direct mass spectrometric methods, such as desorption electrospray ionization (DESI), offer new ways to analyze plant samples directly and rapidly. A strategy for the development and optimization of a DESI method for the direct determination of chemicals on complex surfaces is described. Chlorpropham (CP) was applied to potato surfaces as an example for a crop protection agent and analyzed using a self-made DESI source. Aspects such as instrument selectivity, sensitivity and reproducibility were investigated. The MS4 fragmentation pattern of CP was analyzed to achieve the necessary detection selectivity, and is discussed in detail. Similar fragmentation was found in the ESI and DESI mass spectra, indicating that the mechanisms of ESI and DESI are closely related. A DESI method for semi-quantification of CP on potatoes was developed. Detection limits of 6.5 µg/kg were found using MS/MS. The reproducibility, in the range of 12% (signal variation), appears to be sufficient for semi-quantitative measurements. Copyright © 2013 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

In this article, the electromagnetic (EM) field in gap-mode tip-enhanced Raman spectroscopy (TERS) is investigated theoretically and experimentally for a range of commonly used and unusual metal and nonmetal substrates. By approaching a metal tip to a substrate, both form a coupled system that confines the EM field created at the tip apex. The influence of the substrate onto the EM field enhancement is observed in a top-illumination gap-mode TERS setup for different metal substrates. These include Au, the most commonly used substrate, and also a wide range of rarely or previously unused TERS substrates (Cu, Ag, Al, Pd, Pt, Ni, Ti, Mo, W, stainless steel, Al2O3, SiO2). Self-assembled monolayers of thiols and brilliant cresyl blue thin film samples are investigated experimentally on nine metal substrates, all showing considerable TERS enhancement. With finite difference time domain and finite element simulations used, the article provides a good estimate of the EM field enhancement for a wide range of substrates for users to estimate how well a substrate of choice will perform in a gap-mode TERS experiment. The reduction in EM field strength | E2| compared with Au is less than an order of magnitude for many metals (Calculations: Cu 92%, Ag 81%, Ni 53%). This article experimentally shows that a wide variety of conductive substrates can be used, when one is willing to trade a fraction of the EM field enhancement. TERS was seen on all metal substrates including stainless steel, yet quantification was not always possible. These qualitative results were complemented with intensities from calculations. The wider variety of substrates will increase the applicability of TERS and evolve it one step further towards use in standard analytics. Copyright © 2012 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]