Identification of salivary N-glycoproteins and measurement of glycosylation site occupancy by boronate glycoprotein enrichment and liquid chromatography/electrospray ionization tandem mass spectrometry

RATIONALE Diseases including cancer and congenital disorders of glycosylation have been associated with changes in the site-specific extent of protein glycosylation. Saliva can be non-invasively sampled and is rich in glycoproteins, giving it the potential to be a useful biofluid for the discovery and detection of disease biomarkers associated with changes in glycosylation. METHODS Saliva was collected from healthy individuals and glycoproteins were enriched using phenylboronic acid based glycoprotein enrichment resin. Proteins were deglycosylated with peptide-N-glycosidase F and digested with AspN or trypsin. Desalted peptides and deglycosylated peptides were separated by reversed-phase liquid chromatography and detected with on-line electrospray ionization quadrupole-time-of-flight mass spectrometry using a 5600 TripleTof instrument. Site-specific glycosylation occupancy was semi-quantitatively determined from the abundance of deglycosylated and nonglycosylated versions of each given peptide. RESULTS Glycoprotein enrichment identified 67 independent glycosylation sites from 24 unique proteins, a 3.9-fold increase in the number of glycosylation sites identified. Enrichment of glycoproteins rather than glycopeptides allowed detection of both deglycosylated and nonglycosylated versions of each peptide, and thereby robust measurement of site-specific occupancy at 21 asparagines. Healthy individuals showed limited biological variability in occupancy, with partially modified sites having characteristics consistent with inefficient glycosylation by oligosaccharyltransferase. Inclusion of negative controls without enzymatic deglycosylation controlled for spontaneous chemical deamidation, and identified asparagines previously incorrectly annotated as glycosylated. CONCLUSIONS We developed a sample preparation and mass spectrometry detection strategy for rapid and efficient measurement of site-specific glycosylation occupancy on diverse salivary glycoproteins suitable for biomarker discovery and detection of changes in glycosylation occupancy in human disease.

A high performance liquid chromatography and electrospray ionization mass spectrometry method for the analysis of the natural medicine, Forsythia suspensa

Samples of Forsythia suspensa from raw (Laoqiao) and ripe (Qingqiao) fruit were analyzed with the use of HPLC-DAD and the EIS-MS techniques. Seventeen peaks were detected, and of these, twelve were identified. Most were related to the glucopyranoside molecular fragment. Samples collected from three geographical areas (Shanxi, Henan and Shandong Provinces), were discriminated with the use of hierarchical clustering analysis (HCA), discriminant analysis (DA), and principal component analysis (PCA) models, but only PCA was able to provide further information about the relationships between objects and loadings; eight peaks were related to the provinces of sample origin. The supervised classification models-K-nearest neighbor (KNN), least squares support vector machines (LS-SVM), and counter propagation artificial neural network (CP-ANN) methods, indicated successful classification but KNN produced 100% classification rate. Thus, the fruit were discriminated on the basis of their places of origin.

Photo-ionization and residual electron effects in guided streamers

Complementary experiments and numerical modeling reveal the important role of photo-ionization in the guided streamer propagation in helium-air gas mixtures. It is shown that the minimum electron concentration ∼108 cm−3 is required for the regular, repeated propagation of the plasma bullets, while the streamers propagate in the stochastic mode below this threshold. The stochastic-to-regular mode transition is related to the higher background electron density in front of the propagating streamers. These findings help improving control of guided streamer propagation in applications from health care to nanotechnology and improve understanding of generic pre-breakdown phenomena.

Determination of dimethyl sulfide in gas samples by single photon ionization time of flight mass spectrometry

It is difficult to determine sulfur-containing volatile organic compounds in the atmosphere because of their reactivity. Primary off-line techniques may suffer losses of analytes during the transportation from field to laboratory and sample preparation. In this study, a novel method was developed to directly measure dimethyl sulfide at parts-per-billion concentration levels in the atmosphere using vacuum ultraviolet single photon ionization time-of-flight mass spectrometry. This technique offers continuous sampling at a response rate of one measurement per second, or cumulative measurements over longer time periods. Laboratory prepared samples of different concentrations of dimethyl sulfide in pure nitrogen gas were analyzed at several sampling frequencies. Good precision was achieved using sampling periods of at least 60 seconds with a relative standard deviation of less than 25%. The detection limit for dimethyl sulfide was below the 3 ppb olfactory threshold. These results demonstrate that single photon ionization time-of-flight mass spectrometry is a valuable tool for rapid, real-time measurements of sulfur-containing organic compounds in the air.

Determination of imidacloprid in paddy water and soil by liquid chromatography electrospray ionization-tandem mass spectrometry

A method for the determination of imidacloprid in paddy water and soil was developed using liquid chromatography electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS). Separation of imidacloprid was carried out on a Shimadzu C18 column (150 mm × 4.6 mm, 4.6 μm) with an acetonitrile?water (50 : 50, v/v) mobile phase containing 0.1% of acetic acid. The flow rate was 0.3 mL/min in isocratic mode. The product ion at 209 m/z was selected for quantification in multiple-reaction monitoring scan mode. Imidacloprid residues in soil were extracted by a solid-liquid extraction method with acetonitrile. Water samples were filtered and directly injected for analysis without extraction. Detection limits of 0.5 μg/kg and 0.3 μg/L were achieved for soil and water samples, respectively. The method had recoveries of 90 ± 2% (n = 4) for soil samples and 100 ± 2% (n = 4) for water samples. A linear relationship was observed throughout the investigated range of concentrations (1-200 μg/L), with the correlation coefficients ranging from 0.999 to 1.000. © Pleiades Publishing, Ltd., 2010.

Peripherally η1-Platinated Organometallic Porphyrins as Building Blocks for Multiporphyrin Arrays

The modification of peripherally metalated meso-η1-platiniometalloporphyrins, such as trans-[PtBr(NiDAPP)(PPh3)2] (H2DAPP = 5-phenyl-10,20-bis(3‘,5‘-di-tert-butylphenyl)porphyrin), leads to the analogous platinum(II) nitrato and triflato electrophiles in almost quantitative yields. Self-assembly reactions of these meso-platinioporphyrin tectons with pyridine, 4,4‘-bipyridine, or various meso-4-pyridylporphyrins in chloroform generate new multicomponent organometallic porphyrin arrays containing up to five porphyrin units. These new types of supramolecular arrays are formed exclusively in high yields and are stable in solution or in the solid state for extended periods. They were characterized by multinuclear NMR and UV−visible spectroscopy as well as high-resolution electrospray ionization mass spectrometry.

Differentially Expressed Protein Profile of Human Dental Pulp Cells in the Early Process of Odontoblast-like Differentiation In Vitro

Dental pulp cells (DPCs) are capable of differentiating into odontoblasts that secrete reparative dentin after pulp injury. The molecular mechanisms governing reparative dentinogenesis are yet to be fully understood. Here we investigated the differential protein profile of human DPCs undergoing odontogenic induction for 7 days. Using two-dimensional differential gel electrophoresis coupled with matrix-assisted laser adsorption ionization time of flight mass spectrometry, 2 3 protein spots related to the early odontogenic differentiation were identified. These proteins included cytoskeleton proteins, nuclear proteins, cell membrane-bound molecules, proteins involved in matrix synthesis, and metabolic enzymes. The expression of four identified proteins, which were heteronuclear ribonuclear proteins C, annexin VI, collagen type VI, and matrilin-2, was confirmed by Western blot and real-time realtime polymerase chain reaction analyses. This study generated a proteome reference map during odontoblast- like differentiation of human DPCs, which will be valuable to better understand the underlying molecular mechanisms in odontoblast-like differentiation.

Photoinduced conjugation of Dithioester- and Trithiocarbonate-Functional RAFT polymers with alkenes

We report the photoinduced conjugation of polymers synthesized via reversible addition−fragmentation chain transfer (RAFT) polymerization with a number of low molecular weight (functional) olefins. Upon irradiation of a solution of an aliphatic alkene and the benzyl dithioacetic acid ester (CPDA) or dodecyl trithiocarbonate (DoPAT) functional poly(alkyl acrylate) at the absorption wavelength of the thiocarbonyl group (315 nm), incorporation of the alkene at the polymer chain-end occurred. The most efficient systems identified with regard to the rate of reaction and yield were poly(butyl acrylate)/CPDA/ethyl vinyl ether (78% monoinsertion product after 1 h) and poly(butyl acrylate)/CPDA/1-pentene (73% insertion product after 7 h) at ambient temperature. An in-depth analysis of the reaction mechanism by 1H NMR and online size-exclusion chromatography-electrospray ionization tandem mass spectrometry (SEC/ESI−MSn) revealed that a possible [2 + 2] photoaddition mechanism of conjugation does not take place. Instead, fast β-cleavage of the photoexcited RAFT-end group with subsequent radical addition of an alkene was observed for all employed systems. The presented reaction thus provides a means of spatial and temporal control for the conjugation of alkenes to thiocarbonyl thio-capped macromolecules via the use of UV radiation.

Flexible high voltage pulsed power supply for plasma applications

Demands for delivering high instantaneous power in a compressed form (pulse shape) have widely increased during recent decades. The flexible shapes with variable pulse specifications offered by pulsed power have made it a practical and effective supply method for an extensive range of applications. In particular, the release of basic subatomic particles (i.e. electron, proton and neutron) in an atom (ionization process) and the synthesizing of molecules to form ions or other molecules are among those reactions that necessitate large amount of instantaneous power. In addition to the decomposition process, there have recently been requests for pulsed power in other areas such as in the combination of molecules (i.e. fusion, material joining), gessoes radiations (i.e. electron beams, laser, and radar), explosions (i.e. concrete recycling), wastewater, exhausted gas, and material surface treatments. These pulses are widely employed in the silent discharge process in all types of materials (including gas, fluid and solid); in some cases, to form the plasma and consequently accelerate the associated process. Due to this fast growing demand for pulsed power in industrial and environmental applications, the exigency of having more efficient and flexible pulse modulators is now receiving greater consideration. Sensitive applications, such as plasma fusion and laser guns also require more precisely produced repetitive pulses with a higher quality. Many research studies are being conducted in different areas that need a flexible pulse modulator to vary pulse features to investigate the influence of these variations on the application. In addition, there is the need to prevent the waste of a considerable amount of energy caused by the arc phenomena that frequently occur after the plasma process. The control over power flow during the supply process is a critical skill that enables the pulse supply to halt the supply process at any stage. Different pulse modulators which utilise different accumulation techniques including Marx Generators (MG), Magnetic Pulse Compressors (MPC), Pulse Forming Networks (PFN) and Multistage Blumlein Lines (MBL) are currently employed to supply a wide range of applications. Gas/Magnetic switching technologies (such as spark gap and hydrogen thyratron) have conventionally been used as switching devices in pulse modulator structures because of their high voltage ratings and considerably low rising times. However, they also suffer from serious drawbacks such as, their low efficiency, reliability and repetition rate, and also their short life span. Being bulky, heavy and expensive are the other disadvantages associated with these devices. Recently developed solid-state switching technology is an appropriate substitution for these switching devices due to the benefits they bring to the pulse supplies. Besides being compact, efficient, reasonable and reliable, and having a long life span, their high frequency switching skill allows repetitive operation of pulsed power supply. The main concerns in using solid-state transistors are the voltage rating and the rising time of available switches that, in some cases, cannot satisfy the application’s requirements. However, there are several power electronics configurations and techniques that make solid-state utilisation feasible for high voltage pulse generation. Therefore, the design and development of novel methods and topologies with higher efficiency and flexibility for pulsed power generators have been considered as the main scope of this research work. This aim is pursued through several innovative proposals that can be classified under the following two principal objectives. • To innovate and develop novel solid-state based topologies for pulsed power generation • To improve available technologies that have the potential to accommodate solid-state technology by revising, reconfiguring and adjusting their structure and control algorithms. The quest to distinguish novel topologies for a proper pulsed power production was begun with a deep and through review of conventional pulse generators and useful power electronics topologies. As a result of this study, it appears that efficiency and flexibility are the most significant demands of plasma applications that have not been met by state-of-the-art methods. Many solid-state based configurations were considered and simulated in order to evaluate their potential to be utilised in the pulsed power area. Parts of this literature review are documented in Chapter 1 of this thesis. Current source topologies demonstrate valuable advantages in supplying the loads with capacitive characteristics such as plasma applications. To investigate the influence of switching transients associated with solid-state devices on rise time of pulses, simulation based studies have been undertaken. A variable current source is considered to pump different current levels to a capacitive load, and it was evident that dissimilar dv/dts are produced at the output. Thereby, transient effects on pulse rising time are denied regarding the evidence acquired from this examination. A detailed report of this study is given in Chapter 6 of this thesis. This study inspired the design of a solid-state based topology that take advantage of both current and voltage sources. A series of switch-resistor-capacitor units at the output splits the produced voltage to lower levels, so it can be shared by the switches. A smart but complicated switching strategy is also designed to discharge the residual energy after each supply cycle. To prevent reverse power flow and to reduce the complexity of the control algorithm in this system, the resistors in common paths of units are substituted with diode rectifiers (switch-diode-capacitor). This modification not only gives the feasibility of stopping the load supply process to the supplier at any stage (and consequently saving energy), but also enables the converter to operate in a two-stroke mode with asymmetrical capacitors. The components’ determination and exchanging energy calculations are accomplished with respect to application specifications and demands. Both topologies were simply modelled and simulation studies have been carried out with the simplified models. Experimental assessments were also executed on implemented hardware and the approaches verified the initial analysis. Reports on details of both converters are thoroughly discussed in Chapters 2 and 3 of the thesis. Conventional MGs have been recently modified to use solid-state transistors (i.e. Insulated gate bipolar transistors) instead of magnetic/gas switching devices. Resistive insulators previously used in their structures are substituted by diode rectifiers to adjust MGs for a proper voltage sharing. However, despite utilizing solid-state technology in MGs configurations, further design and control amendments can still be made to achieve an improved performance with fewer components. Considering a number of charging techniques, resonant phenomenon is adopted in a proposal to charge the capacitors. In addition to charging the capacitors at twice the input voltage, triggering switches at the moment at which the conducted current through switches is zero significantly reduces the switching losses. Another configuration is also introduced in this research for Marx topology based on commutation circuits that use a current source to charge the capacitors. According to this design, diode-capacitor units, each including two Marx stages, are connected in cascade through solid-state devices and aggregate the voltages across the capacitors to produce a high voltage pulse. The polarity of voltage across one capacitor in each unit is reversed in an intermediate mode by connecting the commutation circuit to the capacitor. The insulation of input side from load side is provided in this topology by disconnecting the load from the current source during the supply process. Furthermore, the number of required fast switching devices in both designs is reduced to half of the number used in a conventional MG; they are replaced with slower switches (such as Thyristors) that need simpler driving modules. In addition, the contributing switches in discharging paths are decreased to half; this decrease leads to a reduction in conduction losses. Associated models are simulated, and hardware tests are performed to verify the validity of proposed topologies. Chapters 4, 5 and 7 of the thesis present all relevant analysis and approaches according to these topologies.

Challenges in mass spectrometry-based quantification of bioactive peptides: A case study exploring the neuropeptide Y family

The study of biologically active peptides is critical to the understanding of physiological pathways, especially those involved in the development of disease. Historically, the measurement of biologically active endogenous peptides has been undertaken by radioimmunoassay, a highly sensitive and robust technique that permits the detection of physiological concentrations in different biofluid and tissue extracts. Over recent years, a range of mass spectrometric approaches have been applied to peptide quantification with limited degrees of success. Neuropeptide Y (NPY), peptide YY (PYY), and pancreatic polypeptide (PP) belong to the NPY family exhibiting regulatory effects on appetite and feeding behavior. The physiological significance of these peptides depends on their molecular forms and in vivo concentrations systemically and at local sites within tissues. In this report, we describe an approach for quantification of individual peptides within mixtures using high-performance liquid chromatography electrospray ionization tandem mass spectrometry analysis of the NPY family peptides. Aspects of quantification including sample preparation, the use of matrix-matched calibration curves, and internal standards will be discussed. This method for the simultaneous determination of NPY, PYY, and PP was accurate and reproducible but lacks the sensitivity required for measurement of their endogenous concentration in plasma. The advantages of mass spectrometric quantification will be discussed alongside the current obstacles and challenges. © 2012 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 98: 357–366, 2012.

Undrained sediment loading key to long-runout submarine mass movements : evidence from the Caribbean Volcanic Arc

Long undersea debris runout can be facilitated by a boundary layer formed by weak marine sediments under a moving slide mass. Undrained loading of such offshore sediment results in a profound drop of basal shear resistance, compared to subaerial shear resistance, enabling long undersea runout. Thus large long-runout submarine landslides are not truly enigmatic (Voight and Elsworth 1992, 1997), but are understandable in terms of conventional geotechnical principles. A corollary is that remoulded undrained strength, and not friction angle, should be used for basal resistance in numerical simulations. This hypothesis is testable via drilling and examining the structure at the soles of undersea debris avalanches for indications of incorporation of sheared marine sediments, by tests of soil properties, and by simulations. Such considerations of emplacement process are an aim of ongoing research in the Lesser Antilles (Caribbean Sea), where multiple offshore debris avalanche and dome-collapse debris deposits have been identified since 1999 on swath bathymetric surveys collected in five oceanographic cruises. This paper reviews the prehistoric and historic collapses that have occurred offshore of Antilles arc islands and summarizes ongoing research on emplacement processes.

Timing and emplacement dynamics of newly recognised mass flow deposits at ~ 8–12 ka offshore Soufrière Hills volcano, Montserrat : how submarine stratigraphy can complement subaerial eruption histories

This contribution describes two mass movement deposits (total volume ~0.5 km3) identified in seven marine cores located 8 to 15 km offshore southern Montserrat, West Indies. The deposits were emplaced in the last 35 ka and have not previously been recognised in either the subaerial or distal submarine records. Age constraints, provided by radiocarbon dating, show that an explosive volcanic eruption occurred at ca 8–12 ka, emplacing a primary eruption-related deposit that overlies a large (~0.3 km3) reworked bioclastic and volcaniclastic flow deposit, formed from a shelf collapse between 8 and 35 ka. The origin of these deposits has been deduced through the correlation of marine sediment cores, component analysis and geochemical analysis. The 8–12 ka primary volcanic deposit was likely derived from a highly-erosive pyroclastic flow from the Soufrière Hills volcano that entered the ocean and mixed with the water column forming a water-supported density current. Previous investigations of the eruption record suggested that there was a hiatus in activity at the Soufrière Hills volcano between 16 and 6 ka. The ca 8–12 ka eruptive episode identified here shows that this hiatus was shorter than previously hypothesised, and thus highlights the importance of obtaining an accurate and completemarine record of events offshore from volcanic islands and incorporating such data into eruption history reconstructions. Comparisons with the submarine deposit characteristics of the 2003 dome collapse also suggests that the ~8–12 ka eruptive episode was more explosive than eruptions from the current eruptive episode.

Thyroxine and reserpine-induced changes in metabolic profiles of rat urine and the therapeutic effect of Liu Wei Di Huang Wan detected by UPLC-HDMS

The promise of metabonomics, a new "omics" technique, to validate Chinese medicines and the compatibility of Chinese formulas has been appreciated. The present study was undertaken to explore the excretion pattern of low molecular mass metabolites in the male Wistar-derived rat model of kidney yin deficiency induced with thyroxine and reserpine as well as the therapeutic effect of Liu Wei Di Huang Wan (LW) and its separated prescriptions, a classic traditional Chinese medicine formula for treating kidney yin deficiency in China. The study utilized ultra-performance liquid chromatography/electrospray ionization synapt high definition mass spectrometry (UPLC/ESI-SYNAPT-HDMS) in both negative and positive electrospray ionization (ESI). At the same time, blood biochemistry was examined to identify specific changes in the kidney yin deficiency. Distinct changes in the pattern of metabolites, as a result of daily administration of thyroxine and reserpine, were observed by UPLC-HDMS combined with a principal component analysis (PCA). The changes in metabolic profiling were restored to their baseline values after treatment with LW according to the PCA score plots. Altogether, the current metabonomic approach based on UPLC-HDMS and orthogonal projection to latent structures discriminate analysis (OPLS-DA) indicated 20 ions (14 in the negative mode, 8 in the positive mode, and 2 in both) as "differentiating metabolites".

Simultaneous determination by UPLC-ESI-MS of scoparone, capillarisin, rhein, and emodin in rat urine after oral administration of Yin Chen Hao Tang preparation

A simple, sensitive, and validated method was developed for simultaneous determination of scoparone, capillarisin, rhein, and emodin in rat urine by ultra-performance liquid chromatography/electrospray ionization quadruple time-of-flight mass spectrometry (UPLC-MS). The urinary samples were analyzed on an Acquity UPLC BEH C18 1.7 microm 2.1x50 mm column. Scoparone, capillarisin, rhein, and emodin in rat urine were simultaneously analyzed with good separation. The lower limits of detection were 6.0, 9.0, 7.0, and 3.0 ng/mL, and the lower limits of quantification were 20.0, 33.0, 24.0, and 12.0 ng/mL for scoparone, capillarisin, rhein, and emodin, respectively. The intra- and inter-day precisions (RSD) were less than 9%. The intra- and inter-accuracies were found to be in the range of 94.14-104.54% for scoparone, 101.72-107.34% for capillarisin, 95.24-103.59% for rhein, and 101.32-107.82% for emodin at three concentration levels. The absolute recoveries for scoparone, capillarisin, rhein, and emodin were not less than 77.0%. The developed method has been applied to determine scoparone, capillarisin, rhein, and emodin in rat urine after oral administration of Yin Chen Hao Tang preparation, a traditional Chinese medicine formulation widely used in China for treatment of jaundice and liver disorders.

Development and validation of a ultra performance LC-ESI/MS method for analysis of metabolic phenotypes of healthy men in day and night urine samples

Ultra-performance LC coupled to quadrupole TOF/MS (UPLC-QTOF/MS) in positive and negative ESI was developed and validated to analyze metabolite profiles for urine from healthy men during the day and at night. Data analysis using principal components analysis (PCA) revealed differences between metabolic phenotypes of urine in healthy men during the day and at night. Positive ions with mass-to-charge ratio (m/z) 310.24 (5.35 min), 286.24 (4.74 min) and 310.24 (5.63 min) were elevated in the urine from healthy men at night compared to that during the day. Negative ions elevated in day urine samples of healthy men included m/z 167.02 (0.66 min), 263.12 (2.55 min) and 191.03 (0.73 min), whilst ions m/z 212.01 (4.77 min) were at a lower concentration in urine of healthy men during the day compared to that at night. The ions m/z 212.01 (4.77 min), 191.03 (0.73 min) and 310.24 (5.35 min) preliminarily correspond to indoxyl sulfate, citric acid and N-acetylneuraminic acid, providing further support for an involvement of phenotypic difference in urine of healthy men in day and night samples, which may be associated with notably different activities of gut microbiota, velocity of tricarboxylic acid cycle and activity of sialic acid biosynthesis in healthy men as regulated by circadian rhythm of the mammalian bioclock.