Glycosylation modifies the physicochemical properties and protein binding functions of glycoconjugates. These modifications are biosynthesized in the endoplasmic reticulum and Golgi apparatus by a ...series of enzymatic transformations that are under complex control. As a result, mature glycans on a given site are heterogeneous mixtures of glycoforms. This gives rise to a spectrum of adhesive properties that strongly influences interactions with binding partners and resultant biological effects. In order to understand the roles glycosylation plays in normal and disease processes, efficient structural analysis tools are necessary. In the field of glycomics, liquid chromatography/mass spectrometry (LC/MS) is used to profile the glycans present in a given sample. This technology enables comparison of glycan compositions and abundances among different biological samples, i.e. normal versus disease, normal versus mutant, etc. Manual analysis of the glycan profiling LC/MS data is extremely time-consuming and efficient software tools are needed to eliminate this bottleneck. In this work, we have developed a tool to computationally model LC/MS data to enable efficient profiling of glycans. Using LC/MS data deconvoluted by Decon2LS/DeconTools, we built a list of unique neutral masses corresponding to candidate glycan compositions summarized over their various charge states, adducts and range of elution times. Our work aims to provide confident identification of true compounds in complex data sets that are not amenable to manual interpretation. This capability is an essential part of glycomics work flows. We demonstrate this tool, GlycReSoft, using an LC/MS dataset on tissue derived heparan sulfate oligosaccharides. The software, code and a test data set are publically archived under an open source license.
Characterization of the chemical composition and chemical transformations of secondary organic aerosol (SOA) is both a major challenge and the area of greatest uncertainty in current aerosol ...research. This study presents the first application of desorption electrospray ionization combined with high-resolution mass spectrometry (DESI-MS) for detailed chemical characterization and studies of chemical aging of organic aerosol (OA) samples collected on Teflon substrates. DESI-MS offers unique advantages both for detailed characterization of chemically labile components in OA that cannot be detected using traditional electrospray ionization mass spectrometry (ESI-MS) and for studying chemical aging of OA. DESI-MS enables rapid characterization of OA samples collected on substrates by eliminating the sample preparation stage. In addition, it enables detection and structural characterization of chemically labile molecules in OA samples by minimizing the residence time of analyte in the solvent. In this study, DESI-MS and tandem mass spectrometry experiments (MS/MS) were used to examine chemical aging of SOA produced by the ozonolysis of limonene (LSOA) in the presence of gaseous ammonia. Exposure of LSOA to ammonia resulted in measurable changes in the optical properties of the sample observed using ultraviolet (UV)−visible spectroscopy. High-resolution DESI-MS analysis demonstrated that chemical aging results in formation of highly conjugated nitrogen-containing species that are most likely responsible for light-absorbing properties of the aged LSOA. Detailed analysis of the experimental data allowed us to identify several key aging reactions, including the transformation of carbonyls to imines, intramolecular dimerization of imines with other carbonyl compounds in SOA, and intermolecular cyclization of imines. This study presents an important step toward understanding the formation of light-absorbing OA (brown carbon) in the atmosphere.
•This manuscript presents investigation of the critical current dependence of Nb(Ti)N nanostructured superconducting single photon detectors (SNSPD) in function of temperature and applied magnetic ...field.•Presented results are complimentary and compared with the same data received for submicron-wide single bridge Nb(Ti)N structures.•Our data demonstrate significant influence of local constrictions on physical properties of our SNSPD detectors.
Measurements of critical current in NbTiN as a function of applied magnetic field and temperature are reported for two samples: 700-nm-wide bridge and 100-nm-wide meander. In 700-nm-wide NbTiN bridge we pinpointed the limiting factors for the critical current density to be current-driven vortex de-pinning at low temperatures and thermally activated flux flow closer to the transition temperature. In 100-nm-wide NbTiN meander we found phase slips activation, accompanied by hotspots formation at all measured temperatures. These two types of structures demonstrate different dependence of the critical current on the applied magnetic field. Although our NbTiN meander structures has high de-pairing critical current densities ∼107A/cm2 at low temperatures, the real critical currents are smaller due to the presence of the local constrictions.
Microtubules are significant therapeutic targets for the treatment of cancer, where suppression of microtubule dynamicity by drugs such as paclitaxel forms the basis of clinical efficacy. Peloruside ...A, a macrolide isolated from New Zealand marine sponge
Mycale hentscheli, is a microtubule-stabilizing agent that synergizes with taxoid drugs through a unique site and is an attractive lead compound in the development of combination therapies. We report here unique allosteric properties of microtubule stabilization via peloruside A and present a structural model of the peloruside-binding site. Using a strategy involving comparative hydrogen–deuterium exchange mass spectrometry of different microtubule-stabilizing agents, we suggest that taxoid-site ligands epothilone A and docetaxel stabilize microtubules primarily through improved longitudinal interactions centered on the interdimer interface, with no observable contributions from lateral interactions between protofilaments. The mode by which peloruside A achieves microtubule stabilization also involves the interdimer interface, but includes contributions from the α/β-tubulin intradimer interface and protofilament contacts, both in the form of destabilizations. Using data-directed molecular docking simulations, we propose that peloruside A binds within a pocket on the exterior of β-tubulin at a previously unknown ligand site, rather than on α-tubulin as suggested in earlier studies.
Rapid diagnosis of disease states using less invasive, safer, and more clinically acceptable approaches than presently employed is a crucial direction for the field of medicine. While MS-based ...proteomics approaches have attempted to meet these objectives, challenges such as the enormous dynamic range of protein concentrations in clinically relevant biofluid samples coupled with the need to address human biodiversity have slowed their employment. Herein, we report on the use of a new instrumental platform that addresses these challenges by coupling technical advances in rapid gas phase multiplexed ion mobility spectrometry separations with liquid chromatography and MS to dramatically increase measurement sensitivity and throughput, further enabling future high throughput MS-based clinical applications. An initial application of the liquid chromatography - ion mobility spectrometry-MS platform analyzing blood serum samples from 60 postliver transplant patients with recurrent fibrosis progression and 60 nontransplant patients illustrates its potential utility for disease characterization.
Hydrogen/deuterium exchange mass spectrometry (H/DX-MS) experiments implemented to characterize protein interaction and protein folding generate large quantities of data. Organizing, processing and ...visualizing data requires an automated solution, particularly when accommodating new tandem mass spectrometry modes for H/DX measurement. We sought to develop software that offers flexibility in defining workflows so as to support exploratory treatments of H/DX-MS data, with a particular focus on the analysis of very large protein systems and the mining of tandem mass spectrometry data.
We present a software package ("Hydra") that supports both traditional and exploratory treatments of H/DX-MS data. Hydra's software architecture tolerates flexible data analysis procedures by allowing the addition of new algorithms without significant change to the underlying code base. Convenient user interfaces ease the organization of raw data files and input of peptide data. After executing a user-defined workflow, extracted deuterium incorporation values can be visualized in tabular and graphical formats. Hydra also automates the extraction and visualization of deuterium distribution values. Manual validation and assessment of results is aided by an interface that aligns extracted ion chromatograms and mass spectra, while providing a means of rapidly reprocessing the data following manual adjustment. A unique feature of Hydra is the automated processing of tandem mass spectrometry data, demonstrated on a large test data set in which 40,000 deuterium incorporation values were extracted from replicate analysis of approximately 1000 fragment ions in one hour using a typical PC.
The customizable workflows and user-friendly interfaces of Hydra removes a significant bottleneck in processing and visualizing H/DX-MS data and helps the researcher spend more time executing new experiments and interpreting results. This increased efficiency will encourage the analysis of larger protein systems. The ability to accommodate the tandem MS dimension supports alternative data collection and analysis strategies, as well as higher resolution localization of deuteration where permitted by the fragmentation mechanism.
•A new operation regime of NbN superconducting single-photon detectors (SSPDs).•A better understanding of the origin of dark counts generated by the detector.•A promise of PNR functionality in SSPD ...measurements.
We present a new operation regime of NbN superconducting single-photon detectors (SSPDs) by integrating them with a low-noise cryogenic high-electron-mobility transistor and a high-load resistor. The integrated sensors are designed to get a better understanding of the origin of dark counts triggered by the detector, as our scheme allows us to distinguish the origin of dark pulses from the actual photon pulses in SSPDs. The presented approach is based on a statistical analysis of amplitude distributions of recorded trains of the SSPD photoresponse transients. It also enables to obtain information on energy of the incident photons, as well as demonstrates some photon-number-resolving capability of meander-type SSPDs.
The molecular basis of microtubule lattice instability derives from the hydrolysis of GTP to GDP in the lattice-bound state of αβ-tubulin. While this has been appreciated for many years, there is ...ongoing debate over the molecular basis of this instability and the possible role of altered nucleotide occupancy in the induction of a conformational change in tubulin. The debate has organized around seemingly contradictory models. The allosteric model invokes nucleotide-dependent states of curvature in the free tubulin dimer, such that hydrolysis leads to pronounced bending and thus disruption of the lattice. The more recent lattice model describes a predominant role for the lattice in straightening free dimers that are curved regardless of their nucleotide state. In this model, lattice-bound GTP-tubulin provides the necessary force to straighten an incoming dimer. Interestingly, there is evidence for both models. The enduring nature of this debate stems from a lack of high-resolution data on the free dimer. In this study, we have prepared αβ-tubulin samples at high dilution and characterized the nature of nucleotide-induced conformational stability using bottom-up hydrogen/deuterium exchange mass spectrometry (H/DX-MS) coupled with isothermal urea denaturation experiments. These experiments were accompanied by molecular dynamics simulations of the free dimer. We demonstrate an intermediate state unique to GDP-tubulin, suggestive of the curved colchicine-stabilized structure at the intradimer interface but show that intradimer flexibility is an important property of the free dimer regardless of nucleotide occupancy. Our results indicate that the assembly properties of the free dimer may be better described on the basis of this flexibility. A blended model of assembly emerges in which free-dimer allosteric effects retain importance, in an assembly process dominated by lattice-induced effects.
Typical liquid- or gel-based protein separations require enzymatic digestion as an important first step in generating protein identifications. Traditional protocols involve long-term proteolytic ...digestion of the separated protein, often leading to sample loss and reduced sensitivity. Previously, we presented a rapid method of proteolytic digestion that showed excellent digestion of resistant and low concentrations of protein without requiring reduction and alkylation. Here, we demonstrate on-line, real-time tryptic digestion in conjunction with reversed-phase protein separation. The studies were aimed at optimizing pH and ionic strength and the size of the digestion element, to produce maximal protein digestion with minimal effects on chromatographic integrity. Upon establishing optimal conditions, the digestion element was attached downstream from a capillary C4 reversed-phase column. A four-protein mixture was processed through the combined system, and the resulting peptides were analyzed on-line by electrospray mass spectrometry. Extracted ion chromatograms for protein chromatography based on peptide elution were generated. These were shown to emulate ion chromatograms produced in a subsequent run without the digestion element, based on protein elution. The methodology will enable rapid and sensitive analysis of liquid-based protein separations using the power of bottom-up proteomics methodologies.
We present an overview of the state-of-the-art of NbN superconducting single-photon detectors (SSPDs). Our devices exhibit quantum efficiency (QE) of up to 30% in near-infrared wavelength and 0.4% at ...5 mum, with a dark-count rate that can be as low as 10 -4 s -1 . The SSPD structures integrated with lambda/4 microcavities achieve a QE of 60% at telecommunication, 1550-nm wavelength. We have also developed a new generation of SSPDs that possess the QE of large-active-area devices, but, simultaneously, are characterized by low kinetic inductance that allows achieving short response times and the GHz-counting rate with picosecond timing jitter. The improvements presented in the SSPD development, such as fiber-coupled SSPDs, make our detectors most attractive for high-speed quantum communications and quantum computing.
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