Streamlining the Analysis of Proteins from Snake Venom.

Investigating snake venom is necessary for developing new treatments for envenoming and harnessing the therapeutic potential that lies within venom toxins. Despite considerable efforts in previous research, several technical challenges remain for characterizing the individual components within such complex mixtures. Here, we present native and top-down mass spectrometry (MS) workflows that enable the analysis of individual venom proteins within complex mixtures and showcase the utility of these methodologies on King cobra (Ophiophagus hannah) venom. First, we coupled ion mobility spectrometry for separation and electron capture dissociation for charge reduction to resolve highly convoluted mass spectra containing multiple proteins with masses ranging from 55 to 127 kDa. Next, we performed a top-down glycomic analysis of a 25.5 kDa toxin, showing that this protein contains a fucosylated complex glycan. Finally, temperature-controlled nanoelectrospray mass spectrometry facilitated the top-down sequence analysis of a ß-cardiotoxin, which cannot be fragmented by collisional energy due to its disulfide bond pattern. The work presented here demonstrates the applicability of new and promising MS methods for snake venom analysis.

Self-Sorting Collagen Heterotrimers.

Nature uses elaborate methods to control protein assembly, including that of heterotrimeric collagen. Here, we established design principles for the composition and register-selective assembly of synthetic collagen heterotrimers. The assembly code enabled the self-sorting of eight different strands into three─out of 512 possible─triple helices via complementary (4S)-aminoproline and aspartate residues. Native ESI-MS corroborated the specific assembly into coexisting heterotrimers.

Transition Metal Ion FRET-Based Probe to Study Cu(II)-Mediated Amyloid-ß Ligand Binding.

Recent therapeutic strategies suggest that small peptides can act as aggregation inhibitors of monomeric amyloid-ß (Αß) by inducing structural rearrangements upon complexation. However, characterizing the binding events in such dynamic and transient noncovalent complexes, especially in the presence of natively occurring metal ions, remains a challenge. Here, we deploy a combined transition metal ion Förster resonance energy transfer (tmFRET) and native ion mobility-mass spectrometry (IM-MS) approach to characterize the structure of mass- and charge-selected Aß complexes with Cu(II) ions (a quencher) and a potential aggregation inhibitor, a small neuropeptide named leucine enkephalin (LE). We show conformational changes of monomeric Αß species upon Cu(II)-binding, indicating an uncoiled N-terminus and a close interaction between the C-terminus and the central hydrophobic region. Furthermore, we introduce LE labeled at the N-terminus with a metal-chelating agent, nitrilotriacetic acid (NTA). This allows us to employ tmFRET to probe the binding even in low-abundance and transient Aß-inhibitor-metal ion complexes. Complementary intramolecular distance and global shape information from tmFRET and native IM-MS, respectively, confirmed Cu(II) displacement toward the N-terminus of Αß, which discloses the binding region and the inhibitor's orientation.

Rapid Profiling of the Glycosylation Effects on the Binding of SARS-CoV-2 Spike Protein to Angiotensin-Converting Enzyme 2 Using MALDI-MS with High Mass Detection.

The spike protein receptor-binding domain (RBD) of SARS-CoV-2 binds directly to angiotensin-converting enzyme 2 (ACE2), mediating the host cell entry of SARS-CoV-2. Both spike protein and ACE2 are highly glycosylated, which can regulate the binding. Here, we utilized high-mass MALDI-MS with chemical cross-linking for profiling the glycosylation effects on the binding between RBD and ACE2. Overall, it was found that ACE2 glycosylation affects the binding more strongly than does RBD glycosylation. The binding affinity was improved after desialylation or partial deglycosylation (N690) of ACE2, while it decreased after degalactosylation. ACE2 can form dimers in solution, which bind more tightly to the RBD than the ACE2 monomers. The ACE2 dimerization and the binding of RBD to dimeric ACE2 can also be improved by the desialylation or deglycosylation of ACE2. Partial deglycosylation of ACE2 increased the dimerization of ACE2 and the binding affinity of RBD and ACE2 by more than a factor of 2, suggesting its high potential for neutralizing SARS-CoV-2. The method described in the work provided a simple way to analyze the protein-protein interaction without sample purification. It can be widely used for rapid profiling of glycosylation effects on protein-protein interaction for glycosylation-related diseases and the study of multiple interactions between protein and protein aggregates in a single system.

Internal Standard Addition System for Online Breath Analysis.

Breath analysis with secondary electrospray ionization (SESI) coupled to mass spectrometry (MS) is a sensitive method for breath metabolomics. To enable quantitative assessments using SESI-MS, a system was developed to introduce controlled amounts of gases into breath samples and carry out standard addition experiments. The system combines gas standard generation through controlled evaporation, humidification, breath dilution, and standard injection with the help of mass-flow controllers. The system can also dilute breath, which affects the signal of the detected components. This response can be used to filter out contaminating compounds in an untargeted metabolomics workflow. The system's quantitative capabilities have been shown through standard addition of pyridine and butyric acid into breath in real time. This system can improve the quality and robustness of breath data.

Screening Metabolic Biomarkers in KRAS Mutated Mouse Acinar and Human Pancreatic Cancer Cells via Single-Cell Mass Spectrometry.

Pancreatic cancer is a highly aggressive and rapidly progressing disease, often diagnosed in advanced stages due to the absence of early noticeable symptoms. The KRAS mutation is a hallmark of pancreatic cancer, yet the underlying mechanisms driving pancreatic carcinogenesis remain elusive. Cancer cells display significant metabolic heterogeneity, which is relevant to the pathogenesis of cancer. Population measurements may obscure information about the metabolic heterogeneity among cancer cells. Therefore, it is crucial to analyze metabolites at the single-cell level to gain a more comprehensive understanding of metabolic heterogeneity. In this study, we employed a 3D-printed ionization source for metabolite analysis in both mice and human pancreatic cancer cells at the single-cell level. Using advanced machine learning algorithms and mass spectral feature selection, we successfully identified 23 distinct metabolites that are statistically significantly different in KRAS mutant human pancreatic cancer cells and mouse acinar cells bearing the oncogenic KRAS mutation. These metabolites encompass a variety of chemical classes, including organic nitrogen compounds, organic acids and derivatives, organoheterocyclic compounds, benzenoids, and lipids. These findings shed light on the metabolic remodeling associated with KRAS-driven pancreatic cancer initiation and indicate that the identified metabolites hold promise as potential diagnostic markers for early detection in pancreatic cancer patients.

Alternative electrolyte solutions for untargeted breath metabolomics using secondary-electrospray ionization high-resolution mass spectrometry.

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.

Two-dimensional Polymers in Microscopy and Spatially Resolved Vibrational Analysis - A Review.

Two-dimensional polymers comprise a novel class of macromolecules with promising mechanical and chemical properties that have recently become accessible under mild synthetic conditions. As nanometer-sized holes and defects are likely to influence these materials' properties, there is a need for analytical methods to spatially and chemically characterize them down to the nanoscale. In this article, we compare tip-enhanced Raman spectroscopy to common methods in nanoanalysis for the investigation of defect sites in molecularly thin 2D-polymer sheets and briefly present measures to meet the challenges arising from tip-induced degradation processes.

Nanoscale visualization of phase separation in binary supported lipid monolayer using tip-enhanced Raman spectroscopy.

Supported lipid membranes are an important model system to study the phase separation behavior at the nanoscale. However, the conventional nanoanalytical tools often fail to provide reliable chemical characterization of the phase separated domains in a non-destructive and label-free manner. This study demonstrates the application of scanning tunneling microscopy-based tip-enhanced Raman spectroscopy (TERS) to study the nanoscale phase separation in supported d62-DPPC : DOPC lipid monolayers. Hyperspectral TERS imaging successfully revealed a clear segregation of the d62-DPPC-rich and DOPC-rich domains. Interestingly, nanoscale deposits of d62-DPPC were observed inside the DOPC-rich domains and vice versa. High-resolution TERS imaging also revealed the presence of a 40-120 nm wide interfacial region between the d62-DPPC-rich and DOPC-rich domains signifying a smooth transition rather than a sharp boundary between them. The novel insights obtained in this study demonstrate the effectiveness of TERS in studying binary lipid monolayers at the nanoscale.

Systematic study of polymer gas sampling bags for offline analysis of exhaled breath.

Polymeric bags are a widely applied, simple, and cost-effective method for the storage and offline analysis of gaseous samples. Various materials have been used as sampling bags, all known to contain impurities and differing in their cost, durability, and storage capabilities. Herein, we present a comparative study of several well-known bag materials, Tedlar (PVF), Kynar (PVDF), Teflon (PTFE), and Nalophan (PET), as well as a new material, ethylene vinyl copolymer (EVOH), commonly used for storing food. We investigated the influences of storage conditions, humidity, bag cleaning, and light exposure on volatile organic compound concentration (acetone, acetic acid, isoprene, benzene, limonene, among others) in samples of exhaled human breath stored in bags for up to 48 h. Specifically, we show high losses of short-chain fatty acids (SCFAs) in bags of all materials (for most SCFAs, less than 50% after 8 h of storage). We found that samples in Tedlar, Nalophan, and EVOH bags undergo changes in composition when exposed to UV radiation over a period of 48 h. We report high initial impurity levels in all the bags and their doubling after a period of 48 h. We compare secondary electrospray ionization and proton transfer reaction mass spectrometry in the context of offline analysis after storage in sampling bags. We provide an analytical perspective on the temporal evolution of bag contents by presenting the intensity changes of all significantm/zfeatures. We also present a simple, automated, and cost-effective offline sample introduction system, which enables controlled delivery of collected gaseous samples from polymeric bags into the mass spectrometer. Overall, our findings suggest that sampling bags exhibit high levels of impurities, are sensitive to several environmental factors (e.g. light exposure), and provide low recoveries for some classes of compounds, e.g. SCFAs.

Nanoscale Chemical Analysis of Thin Film Solar Cell Interfaces Using Tip-Enhanced Raman Spectroscopy.

Interfacial regions play a key role in determining the overall power conversion efficiency of thin film solar cells. However, the nanoscale investigation of thin film interfaces using conventional analytical tools is challenging due to a lack of required sensitivity and spatial resolution. Here, we surmount these obstacles using tip-enhanced Raman spectroscopy (TERS) and apply it to investigate the absorber (Sb2Se3) and buffer (CdS) layers interface in a Sb2Se3-based thin film solar cell. Hyperspectral TERS imaging with 10 nm spatial resolution reveals that the investigated interface between the absorber and buffer layers is far from uniform, as TERS analysis detects an intermixing of chemical compounds instead of a sharp demarcation between the CdS and Sb2Se3 layers. Intriguingly, this interface, comprising both Sb2Se3 and CdS compounds, exhibits an unexpectedly large thickness of 295 ± 70 nm attributable to the roughness of the Sb2Se3 layer. Furthermore, TERS measurements provide compelling evidence of CdS penetration into the Sb2Se3 layer, likely resulting from unwanted reactions on the absorber surface during chemical bath deposition. Notably, the coexistence of ZnO, which serves as the uppermost conducting layer, and CdS within the Sb2Se3-rich region has been experimentally confirmed for the first time. This study underscores TERS as a promising nanoscale technique to investigate thin film inorganic solar cell interfaces, offering novel insights into intricate interface structures and compound intermixing.

Probing the Stability of a ß-Hairpin Scaffold after Desolvation.

Probing the structural characteristics of biomolecular ions in the gas phase following native mass spectrometry (nMS) is of great interest, because noncovalent interactions, and thus native fold features, are believed to be largely retained upon desolvation. However, the conformation usually depends heavily on the charge state of the species investigated. In this study, we combine transition metal ion Förster resonance energy transfer (tmFRET) and ion mobility-mass spectrometry (IM-MS) with molecular dynamics (MD) simulations to interrogate the ß-hairpin structure of GB1p in vacuo. Fluorescence lifetime values and collisional cross sections suggest an unfolding of the ß-hairpin motif for higher charge states. MD simulations are consistent with experimental constraints, yet intriguingly provide an alternative structural interpretation: preservation of the ß-hairpin is not only predicted for 2+ but also for 4+ charged species, which is unexpected given the substantial Coulomb repulsion for small secondary structure scaffolds.

Uncoupling of behavioral and metabolic 24-h rhythms in reindeer.

Reindeer in the Arctic seasonally suppress daily circadian patterns of behavior present in most animals.1 In humans and mice, even when all daily behavioral and environmental influences are artificially suppressed, robust endogenous rhythms of metabolism governed by the circadian clock persist and are essential to health.2,3 Disrupted rhythms foster metabolic disorders and weight gain.4 To understand circadian metabolic organization in reindeer, we performed behavioral measurements and untargeted metabolomics from blood plasma samples taken from Eurasian tundra reindeer (Rangifer tarandus tarandus) across 24 h at 2-h intervals in four seasons. Our study confirmed the absence of circadian rhythms of behavior under constant darkness in the Arctic winter and constant daylight in the Arctic summer, as reported by others.1 We detected and measured the intensity of 893 metabolic features in all plasma samples using untargeted ultra-high-performance liquid chromatography-mass spectrometry (UPLC-MS). A core group of metabolites (66/893 metabolic features) consistently displayed 24-h rhythmicity. Most metabolites displayed a robust 24-h rhythm in winter and spring but were arrhythmic in summer and fall. Half of all measured metabolites displayed ultradian sleep-wake dependence in summer. Irrespective of the arrhythmic behavior, metabolism is rhythmic (24 h) in seasons of low food availability, potentially favoring energy efficiency. In seasons of food abundance, 24-h rhythmicity in metabolism is drastically reduced, again irrespective of behavioral rhythms, potentially fostering weight gain.

Stoichiometry and architecture of the human pyruvate dehydrogenase complex.

The pyruvate dehydrogenase complex (PDHc) is a key megaenzyme linking glycolysis with the citric acid cycle. In mammalian PDHc, dihydrolipoamide acetyltransferase (E2) and the dihydrolipoamide dehydrogenase-binding protein (E3BP) form a 60-subunit core that associates with the peripheral subunits pyruvate dehydrogenase (E1) and dihydrolipoamide dehydrogenase (E3). The structure and stoichiometry of the fully assembled, mammalian PDHc or its core remained elusive. Here, we demonstrate that the human PDHc core is formed by 48 E2 copies that bind 48 E1 heterotetramers and 12 E3BP copies that bind 12 E3 homodimers. Cryo-electron microscopy, together with native and cross-linking mass spectrometry, confirmed a core model in which 8 E2 homotrimers and 12 E2-E2-E3BP heterotrimers assemble into a pseudoicosahedral particle such that the 12 E3BP molecules form six E3BP-E3BP intertrimer interfaces distributed tetrahedrally within the 60-subunit core. The even distribution of E3 subunits in the peripheral shell of PDHc guarantees maximum enzymatic activity of the megaenzyme.

Deterministic grayscale nanotopography to engineer mobilities in strained MoS2 FETs.

Field-effect transistors (FETs) based on two-dimensional materials (2DMs) with atomically thin channels have emerged as a promising platform for beyond-silicon electronics. However, low carrier mobility in 2DM transistors driven by phonon scattering remains a critical challenge. To address this issue, we propose the controlled introduction of localized tensile strain as an effective means to inhibit electron-phonon scattering in 2DM. Strain is achieved by conformally adhering the 2DM via van der Waals forces to a dielectric layer previously nanoengineered with a gray-tone topography. Our results show that monolayer MoS2 FETs under tensile strain achieve an 8-fold increase in on-state current, reaching mobilities of 185 cm²/Vs at room temperature, in good agreement with theoretical calculations. The present work on nanotopographic grayscale surface engineering and the use of high-quality dielectric materials has the potential to find application in the nanofabrication of photonic and nanoelectronic devices.