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When characterizing components from ginseng, we found a vast number of multicharged anions presented in the liquid chromatography-mass spectrometry (LC-MS) chromatograms. The source of these anions is unclear yet, while ginsenosides, the major components of ginseng, are the main suspected type of molecules because of their sugar moiety. Our investigation using 14 pure ginsenosides affirmed that the multicharged anions were formed by ginsenosides rather than other types of ingredients in ginseng. Various anions could be observed for each ginsenoside. These anions contain ions ([M-2H]2−, [M+Adduct]2−), as well as those formed by polymerization of at least two ginsenosides, such as [nM-2H]2−, [nM-H+Adduct]2−, and [nM-3H]3−. The presence of so different types of ions from a ginsenoside explains the reason for the large number of anions in the LC-MS analysis of ginseng. We further found that formation of [nM-2H]2− ions was influenced by the number of sugar chains: ginsenosides containing two sugar chains produced all [nM-2H]2− ion types, whereas ginsenosides containing one sugar chain did not produce [2M-2H]2−. Thus, [2M-2H]2− and [3M-2H]2− can be utilized to rapidly identify monodesmosidic and/or bidesmosidic ginsenosides as joint diagnostic anions. The position of the glycosyl radical might be the key factor affecting the formation of multicharged multimer ions from monodesmosidic ginsenosides. Consequently, three groups of ginsenoside isomers were differentiated by characteristic [nM-2H]2− anions. Using concentration-dependent characteristics and collision-induced dissociation (CID), we confirmed that [nM-2H]2− ions are non-covalently bound multimers whose aggregation has marked distinction between monodesmosidic and bidesmosidic ginsenosides, accounting for the differentiated formation of [nM-2H]2− between them.Graphical Abstract [ABSTRACT FROM AUTHOR]

Evidence for the existence of condensed-phase isomers of silver-lipoate clusters, Ag29(LA)12, where LA = (R)-a lipoic acid, was obtained by reversed-phase ion-pair liquid chromatography with in-line UV-vis and electro-spray ionization (ESI)-MS detection. All components of a raw mixture were separated according to surface chemistry and increasing size via reversed-phase gradient HPLC methods and identified by their corresponding m/z ratio by ESI in the negative ionization mode. Aqueous and methanol mobile-phase mixtures, each containing 400 mM hexafluoroisopropanol (HFIP)-15 mM triethylamine (TEA), were employed to facilitate the interaction between the clusters and stationary phase via formation of ion-pairs. TEA-HFIP (triethylammonium-hexafluoroisoprop-oxide) had been shown to provide superior chromatographic peak shape and mass spectral signal compared with alternative modifiers such as TEAA (triethylammo-nium-acetate) for analysis of oligonucleotide samples. Liquid chromatographic separation prior to mass spectrometry detection facilitated sample analysis by production of simplified mass spectra for each eluting cluster species and provided insight into the existence of at least two major solution-phase isomers of Ag29(LA)12. UV-vis detection in-line with ESI analysis provided independent confirmation of the existence of the isomers and their similar electronic structure as judged from their identical optical spectra in the 300-500 nm range. [Diastereomerism provides a possible interpretation for the near-equal abundance of the two forms, based on a structurally defined nonaqueous homologue.] [ABSTRACT FROM AUTHOR]

ABSTRACT: Replacement of capillary with solid substrates for sample loading and ionization has created many new possibilities for electrospray ionization mass spectrometry (ESI-MS). Surface modification is an attractive strategy to enhance the analytical capability of solid-substrate ESI-MS and allow understanding the relationship between surface activity of solid substrates and analytical properties. In this study, we performed surface modification of wooden tips with hydrophobic (-C18), basic (-NH2), and acidic (-SO3H) functional groups and applied various sampling methods, i.e., extractive sampling and direct loading, to comprehensively investigate the analytical properties of solid-substrate ESI-MS. Our results showed that, for the direct loading method, analytes with weak interactions with solid-substrate surface could be readily sprayed out for detection. While for the extractive sampling method, analytes strongly retained on solid-substrate surface could be selectively enriched and detected, and a washing step after sample loading could effectively remove unbound components for reducing interference. Overall, the insights on the effects of surface -- analyte interactions on the analytical features obtained in this study could aid the development of surface-modified strategies for enhancing the analytical capability of solid-substrate ESI-MS. [ABSTRACT FROM AUTHOR]

Electrospray ionization (ESI) is widely used in liquid chromatography coupled to mass spectrometry (LC-MS) for the analysis of biomolecules. However, the ESI process is still not completely understood, and it is often a matter of trial and error to enhance ESI efficiency and, hence, the response of a given set of compounds. In this work we performed a systematic study of the ESI response of 14 amino acids that were acylated with organic acid anhydrides of increasing chain length and with poly(ethylene glycol) (PEG) changing certain physicochemical properties in a predictable manner. By comparing the ESI response of 70 derivatives, we found that there was a strong correlation between the calculated molecular volume and the ESI response, while correlation with hydrophobicity (log P values), pK[sub a], and the inverse calculated surface tension was significantly lower although still present, especially for individual derivatized amino acids with increasing acyl chain lengths. Acylation with PEG containing five ethylene glycol units led to the largest gain in ESI response. This response was maximal independent of the calculated physicochemical properties or the type of amino acid. Since no actual physicochemical data is available for most derivatized compounds, the responses were also used as input for a quantitative structure-property relationship (QSPR) model to find the best physicochemical descriptors relating to the ESI response from molecular structures using the amino acids and their derivatives as a reference set. A topological descriptor related to molecular size (SPAN) was isolated next to a descriptor related to the atomic composition and structural groups (BIC0). The validity of the model was checked with a test set of 43 additional compounds that were unrelated to amino acids. While prediction was generally good (R² > 0.9), compounds containing halogen atoms or nitro groups gave a lower predicted ESI response. [ABSTRACT FROM AUTHOR]

We describe the DRILL (dry ion localization and locomotion) device, which is an interface for electrospray ionization (ESI)-mass spectrometry (MS) that exploits a swirling flow to enable the use of inertial separation to prescribe different fates for electrosprayed droplets based on their size. This source adds a new approach to charged droplet trajectory manipulation which, when combined with hydrodynamic drag forces and electric field forces, provides a rich range of possible DRILL operational modes. Here, we experimentally demonstrate sensitivity improvement obtained via vortex-induced inertial sorting of electrosprayed droplets/ions: one possible mode of DRILL operation. In this mode, DRILL removes larger droplets while accelerating the remainder of the ESI plume, producing a high velocity stream of gas-enriched spray with small, highly charged droplets and ions and directing it toward the MS inlet. The improved signal-to-noise ratio (10-fold enhancement) in the detection of angiotensin I is demonstrated using the DRILL interface coupled to ESI-MS along with an improved limit of detection (10-fold enhancement, 100 picomole) in the detection of angiotensin II. The utility of DRILL has also been demonstrated by liquid chromatography (LC)-MS: a stable isotope labeled peptide cocktail was spiked into a complex native tissue extract and quantified by unscheduled multiple reaction monitoring on a TSQ Vantage. DRILL demonstrated improved signal strength (up to a 700-fold) for 8 out of 9 peptides and had no effects on the peak shape of the transitions. [ABSTRACT FROM AUTHOR]

Efforts to analyze trace levels of cyclic peroxides by liquid chromatography/mass spectrometry gave evidence that acetonitrile suppressed ion formation. Further investigations extended this discovery to ketones, linear peroxides, esters, and possibly many other types of compounds, including triazole and menadione. Direct ionization suppression caused by acetonitrile was observed for multiple adduct types in both electrospray ionization and atmospheric pressure chemical ionization. The addition of only 2% acetonitrile significantly decreased the sensitivity of analyte response. Efforts to identify the mechanism were made using various nitriles. The ion suppression was reduced by substitution of an acetonitrile hydrogen with an electron-withdrawing group, but was exacerbated by electron-donating or steric groups adjacent to the nitrile. Although current theory does not explain this phenomenon, we propose that polar interactions between the various functionalities and the nitrile may be forming neutral aggregates that manifest as ionization suppression. [ABSTRACT FROM AUTHOR]

Direct analysis of microbial cocultures grown on agar media by desorption electrospray ionization mass spectrometry (DESI-MS) is quite challenging. Due to the high gas pressure upon impact with the surface, the desorption mechanism does not allow direct imaging of soft or irregular surfaces. The divots in the agar, created by the high-pressure gas and spray, dramatically change the geometry of the system decreasing the intensity of the signal. In order to overcome this limitation, an imprinting step, in which the chemicals are initially transferred to flat hard surfaces, was coupled to DESI-MS and applied for the first time to fungal cocultures. Note that fungal cocultures are often disadvantageous in direct imaging mass spectrometry. Agar plates of fungi present a complex topography due to the simultaneous presence of dynamic mycelia and spores. One of the most devastating diseases of cocoa trees is caused by fungal phytopathogen Moniliophthora roreri. Strategies for pest management include the application of endophytic fungi, such as Trichoderma harzianum, that act as biocontrol agents by antagonizing M. roreri. However, the complex chemical communication underlying the basis for this phytopathogen-dependent biocontrol is still unknown. In this study, we investigated the metabolic exchange that takes place during the antagonistic interaction between M. roreri and T. harzianum. Using imprint-DESI-MS imaging we annotated the secondary metabolites released when T. harzianum and M. roreri were cultured in isolation and compared these to those produced after 3 weeks of coculture. We identified and localized four phytopathogen-dependent secondary metabolites, including T39 butenolide, harzianolide, and sorbicillinol. In order to verify the reliability of the imprint-DESI-MS imaging data and evaluate the capability of tape imprints to extract fungal metabolites while maintaining their localization, six representative plugs along the entire M. roreri/T. harzianum coculture plate were removed, weighed, extracted, and analyzed by liquid chromatography-high-resolution mass spectrometry (LC-HRMS). Our results not only provide a better understanding of M. roreri-dependent metabolic induction in T. harzianum, but may seed novel directions for the advancement of phytopathogen-dependent biocontrol, including the generation of optimized Trichoderma strains against M. roreri, new biopesticides, and biofertilizers. [ABSTRACT FROM AUTHOR]

A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method is described that employs a novel derivatization reagent for the measurement of serum estradiol (E2), with simultaneous analysis of underivatized testosterone (T) and dihydrotestosterone (DHT). The main advantage of the new derivatization reagent 1,2-dimethylimidazole-5-sulfonyl chloride is its analyte-specific fragmentation that enables monitoring of confirmatory mass transitions with high sensitivity. The reaction mixture can be analyzed without additional purification steps using a 9.5 min gradient run, and sensitive detection is achieved with a triple quadrupole mass spectrometer using atmospheric pressure photoionization. Method validation was performed with human serum samples, including a comparison with a standard LC-MS/MS method using 120 samples from a clinical study, and analysis of certified E2 serum reference materials BCR-576, BCR-577, and BCR-578. The lower limits of quantification for E2, T, and DHT were 0.5 pg/mL, 25 pg/mL, and 0.10 ng/mL, respectively, from a 200-μL sample. Validation results indicated good accuracy and agreement with established, conventional LC-MS/MS assays, demonstrating suitability for analysis of samples containing E2 in the low pg/mL range, such as serum from men, children, and postmenopausal women. [ABSTRACT FROM AUTHOR]

A simple to construct and operate, "dip-in" electromembrane extraction (EME) probe directly coupled to electrospray ionization-mass spectrometry (ESI-MS) for rapid extraction and real time analysis of various analytes was developed. The setup demonstrated that EME-MS can be used as a viable alternative to conventional protein precipitation followed by liquid chromatography–mass spectrometry (LC–MS) for studying drug metabolism. Comparison of EME-MS with LC–MS for drug metabolism analysis demonstrated for the first time that real time extraction of analytes by EME is possible. Metabolism kinetics were investigated for three different drugs: amitriptyline, promethazine, and methadone. By comparing the EME-MS extraction profiles of the drug substances and formed drug metabolites with the metabolism profiles obtained by conventional protein precipitation followed by LC–MS good correlation was obtained with only very limited time delay in the extraction. The results indicate that, by tuning the electromembrane properties, for example, by optimizing the extraction voltage, extremely fast extraction kinetics can be obtained. A metabolic profile could be generated while the drug was metabolized offering a significant time saving as compared to conventional LC–MS where laborious protein precipitation or other sample pretreatments are required before analysis. This makes the developed EME-MS setup a highly promising sample preparation method for various kinds of applications where fast and real-time analysis of analytes is of interest. [ABSTRACT FROM AUTHOR]

A simple and reliable liquid chromatography-mass spectrometry (LC-MS) assay has been developed and validated for the kinetic characterization and evaluation of inhibitors of shikimate kinase from Mycobacterium tuberculosis (MtSK), a potential target for the development of novel antitubercular drugs. This assay is based on the direct determination of the reaction product shikimate-3-phosphate (S3P) using electrospray ionization (ESI) and a quadrupole time-of-flight (Q-TOF) detector. A comparative analysis of the kinetic parameters of MtSK obtained by the LC-MS assay with those obtained by a conventional UV-assay was performed. Kinetic parameters determined by LC-MS were in excellent agreement with those obtained from the UV assay, demonstrating the accuracy, and reliability of this method. The validated assay was successfully applied to the kinetic characterization of a known inhibitor of shikimate kinase; inhibition constants and mode of inhibition were accurately delineated with LC-MS. [ABSTRACT FROM AUTHOR]

Global urine metabolomics is a rapidly expanding field with the potential to discover biomarkers of disease and exposure. To date, most methods focus on rapid sample preparation, using neat or diluted urine, together with high-throughput analyses, and are poorly suited for detection of low abundance metabolites present in urine samples. In this study, novel methods have been developed to analyze urine by splitless nanoflowUHPLC-nanoESI-TOFMS (nUHPLC-nESI-TOFMS) after preconcentration by solid-phase extraction (SPE), thus enabling significant improvements in analytical sensitivity and coverage of the urinary metabolome. In initial work, urine samples were extracted by both anion and cation exchange mixed-mode polymeric SPE cartridges and qualitatively compared with those using conventional sample preparations using UHPLC-ESI-TOFMS analyses. Compared with neat or diluted urine samples, SPE concentration of urine resulted in detection of additional metabolites including bile acids, lipids, pharmaceuticals, and markers of lifestyle, with little loss of other components of the metabolome. Analyses of SPE preparations by nUHPLC-nESI-TOFMS revealed excellent retention time repeatability with <1% coefficient of variation (CV) for 96% of analyzed peaks. The repeatability of the MS response was <30% CV for >79% of MS features in both negative and positive nESI modes. Compared with UHPLC-ESI-TOFMS, analysis by the nanoplatform enabled detection of signaling molecules important in disease processes including sex steroids, glucocorticoids, eicosanoids, and neurotransmitter metabolites. The significant improvement in sensitivity arising from use of splitless nUHPLC-nESI-TOFMS analyses of SPE-concentrated samples represents a step change in coverage of the urinary metabolome, thereby increasing the potential for biomarker discovery. [ABSTRACT FROM AUTHOR]

Low molecular mass (LMM) thiols is a diverse group of compounds, which play several important roles in aquatic ecosystems, even though they typically occur at low concentrations. Comprehensive studies of LMM thiols in natural waters have so far been hampered by selectivity and limit of detection constraints of previous analytical methods. Here, we describe a selective and robust method for the quantification of 16 LMM thiols in natural waters. Thiols were derivatized with 4-(hydroxymercuri)benzoate (PHMB) and preconcentrated online by solid-phase extraction (SPE) before separation by liquid chromatography and determination by electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). Their quantification was performed by selective reaction monitoring (SRM), while the presence of a product ion at m/z 355, specific for thiols and common for the investigated compounds, also allows to screen samples for unknown thiols by a precursor ion scan approach. The robustness of the method was validated for aqueous matrices with different pH, sulfide, and dissolved organic carbon (DOC) concentrations. The limits of detection for the thiols were in the sub-nanomolar range (0.06-0.5 nM) and the methodology allowed determination of both reduced and total thiol concentrations (using tris(2-carboxyethyl)phosphine (TCEP) as reducing agent). Six thiols (mercaptoacetic acid, cysteine, homocysteine, N-acetyl-cysteine, mercaptoethane-sulfonate, and glutathione) were detected with total concentrations of 7-153 nM in boreal lake or wetland pore waters while four thiols (mercaptoacetic acid, cysteine, homocysteine, and N-acetyl-cysteine) were detected in their reduced form at concentrations of 5-80 nM. [ABSTRACT FROM AUTHOR]

This manuscript reports a new pesticide residue analysis method employing a microflow-liquid chromatography system coupled to a triple quadrupole mass spectrometer (microflow-LC-ESI-QqQ-MS). This uses an electrospray ionization source with a narrow tip emitter to generate smaller droplets. A validation study was undertaken to establish performance characteristics for this new approach on 90 pesticide residues, including their degradation products, in three commodities (tomato, pepper, and orange). The significant benefits of the microflow-LC-MS/MS-based method were a high sensitivity gain and a notable reduction in matrix effects delivered by a dilution of the sample (up to 30-fold); this is as a result of competition reduction between the matrix compounds and analytes for charge during ionization. Overall robustness and a capability to withstand long analytical runs using the microflow-LC-MS system have been demonstrated (for 100 consecutive injections without any maintenance being required). Quality controls based on the results of internal standards added at the samples' extraction, dilution, and injection steps were also satisfactory. The LOQ values were mostly 5 μg kg-1 for almost all pesticide residues. Other benefits were a substantial reduction in solvent usage and waste disposal as well as a decrease in the run-time. The method was successfully applied in the routine analysis of 50 fruit and vegetable samples labeled as organically produced. [ABSTRACT FROM AUTHOR]

Microstructured fibers (MSFs) having raised polymer nozzles in each channel are custom designed, fabricated, and tested for use as multiple electrospray (MES) emitters for mass spectrometry (MS). There is strong motivation to develop electrospray emitters that operate at practical flow rates but give the much greater ionization efficiency associated with lower (nano) flow rates. This can be accomplished by splitting the flow into many lower-volume electrosprays, an approach known as MES. To couple with most modern mass spectrometers, the MES emitter must have a small diameter to allow efficient ion collection into the MS. In this work, a MSF, defined as a fiber having many empty channels running along its length, was designed to have 9 channels, 9 μm each, >100 μm apart arranged in a radial pattern, all in a fiber having a compatible diameter with both front-end LC equipment and typical MS inlets. This design seeks to promote independent electrospray from each channel while maintaining electric field homogeneity. While the MSFs themselves do not support MES, the formation of polymer nozzles protruding from each channel at the tip face enables independent electrospray from each nozzle. Microscope imaging, electrospray current measurement, and ESI-MS detection of a model analyte all confirm the MES behavior of the 9-nozzle emitter, showing significant signal enhancement relative to a single-nozzle emitter at the same total flow rate. LC/MS data from a protein digest obtained at an independent laboratory demonstrates the applicability and robustness of the emitter for real scientific challenges using modern LC/MS equipment. [ABSTRACT FROM AUTHOR]

This study presents on-tissue proteolytic digestion using a microwave irradiation and peptide extraction method for in situ analysis of proteins from spatially defined regions of a tissue section. The methodology utilizes hydrogel discs (1 mm diameter) embedded with trypsin solution. The enzyme-laced hydrogel discs are applied to a tissue section, directing enzymatic digestion to a spatially confined area of the tissue. By applying microwave radiation, protein digestion is performed in 2 min on-tissue, and the extracted peptides are then analyzed by matrix assisted laser desorption/ionization mass spectrometry (MALDI MS) and liquid chromatography tandem mass spectrometry (LC-MS/MS). The reliability and reproducibility of the microwave assisted hydrogel mediated on-tissue digestion is demonstrated by the comparison with other on-tissue digestion strategies, including comparisons with conventional heating and in-solution digestion. LC-MS/MS data were evaluated considering the number of identified proteins as well as the number of protein groups and distinct peptides. The results of this study demonstrate that rapid and reliable protein digestion can be performed on a single thin tissue section while preserving the relationship between the molecular information obtained and the tissue architecture, and the resulting peptides can be extracted in sufficient abundance to permit analysis using LC-MS/MS. This approach will be most useful for samples that have limited availability but are needed for multiple analyses, especially for the correlation of proteomics data with histology and immunohistochemistry. [ABSTRACT FROM AUTHOR]

In the present work, direct analysis of real time ionization combined with multi-stage tandem mass spectrometry (DART-MS) was used to investigate the metabolic profile of aconite alkaloids in rat intestinal bacteria. A total of 36 metabolites from three aconite alkaloids were identified by using DART-MS, and the feasibility of quantitative analysis of these analytes was examined. Key parameters of the DART ion source, such as helium gas temperature and pressure, the source-to-MS distance, and the speed of the autosampler, were optimized to achieve high sensitivity, enhance reproducibility, and reduce the occurrence of fragmentation. The instrument analysis time for one sample can be less than 10 s for this method. Compared with ESI-MS and UPLC-MS, the DART-MS is more efficient for directly detecting metabolic samples, and has the advantage of being a simple, high-speed, high-throughput method. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

Subambient pressure ionization with nanoelectrospray (SPIN) has proven to be effective in producing ions with high efficiency and transmitting them to low pressures for increased sensitivity in mass spectrometry (MS) analysis. Here we present evidence that the SPIN source not only improves MS sensitivity but also facilitates the detection of more labile compounds. The gentleness of conventional heated capillary electrospray ionization (ESI) and the SPIN designs was compared in conjunction with the liquid chromatography mass spectrometry (LC-MS) analysis of colominic acid and N-glycans containing sialic acid. Prior experiments conducted with the SPIN interface demonstrated the ability to detect labile glycans such as heavily sialylated and polysialic acid N-glycans, which are difficult to detect with a conventional ESI-MS interface. Colominic acid is a mixture of sialic acid polymers of different lengths containing labile glycosidic linkages between monomer units necessitating a gentle ion source. These labile covalent bonds may display similar behavior to sialic acid chains in N-glycans during MS analysis. By coupling the SPIN source with high-resolution mass spectrometry and using advanced data processing tools, we demonstrate much extended coverage of sialic acid polymer chains as compared to conventional ESI-MS and the ability to detect sialic acid containing N-glycans without the need of sample derivatization. In addition, we show that SPIN-LC-MS is effective in elucidating polymer features with highefficiency and high sensitivity previously unattainable by the conventional ESI-LC-MS methods. [ABSTRACT FROM AUTHOR]

An automated analyte electroextraction and preconcentration system, which was used as the front end for a liquid chromatography-electrospray mass spectrometry instrument, is described. The extraction was based on driving the anionic analytes across a polymer inclusion membrane by application of a potential of 200 V to the cell. Five milliliters (5 mL) of sample were passed through a flow-through cell at a flow rate of 0.2 mL/min containing a membrane 20 μm thick. This consisted of 75% cellulose triacetate as base polymer, 12.5% of tris(2-ethylhexyl)phosphate as plasticizer, and 12.5% of Aliquat 336 as cationic carrier. The target analytes were enriched in 20 μL of a stagnant acceptor solution prior to online LC/MS analysis. The performance of the system was demonstrated for the determination of chlorinated phenoxyacetic acid herbicides in spiked river water. Enrichment factors of ~200 were achieved with recoveries of typically 99% and precision values of typically 5%. The limit of detection (LOD) values were found to be between 0.03 ng/mL to 0.08 ng/mL. [ABSTRACT FROM AUTHOR]

The orange carotenoid protein (OCP) plays a photoprotective role in cyanobacterial photosynthesis similar to that of nonphotochemical quenching in higher plants. Under high-light conditions, the OCP binds to the phycobilisome (PBS) and reduces the extent of transfer of energy to the photosystems. The protective cycle starts from a light-induced activation of the OCP. Detailed information about the molecular mechanism of this process as well as the subsequent recruitment of the active OCP to the phycobilisome are not known. We report here our investigation on the OCP photoactivation from the cyanobacterium Synechocystis sp. PCC 6803 by using a combination or native electrospray mass spectrometry (MS) and protein cross-linking. We demonstrate that native MS can capture the OCP with its intact pigment and further reveal that the OCP undergoes a dimer-to-monomer transition upon light illumination. The reversion of the activated form of the OCP to the inactive, dark form was also observed by using native MS. Furthermore, in vitro reconstitution of the OCP and PBS allowed us to perform protein chemical cross-linking experiments. Liquid chromatography-MS/MS analysis identified cross-linking species between the OCP and the PBS core components. Our result indicates that the N-terminal domain of the OCP is closely involved in the association with a site formed by two allophycocyanin trimers in the basal cylinders of the phycobilisome core. This report improves our understanding of the activation mechanism of the OCP and the structural binding site of the OCP during the cyanobacterial nonphotochemical quenching process. [ABSTRACT FROM AUTHOR]

We report a chemical-vapor-assisted electrospray ionization (ESI) technique to improve the detection sensitivity of ESI mass spectrometry (MS). This simple technique involves introducing a chemical vapor into the sheath gas around the nano-ESI spray tip or through a tubing with its outlet placed close to the spray tip. A variety of chemical vapors were tested and found to have varying degrees of effects on analyte signal intensities. The use of benzyl alcohol vapors in ESI was found to increase signal intensities of standard peptides by up to 4-fold. When this technique was combined with capillary liquid chromatography tandem MS (LC-MS/MS), the number of unique peptides identified in the acid hydrolysate of alpha casein increased by 45% and the number of peptides and proteins identified in a tryptic digest of E. coli cell lysate increased by 13% and 14%, respectively, along with an increased average match score. This technique could also increase the analyte signals for some small molecules, such as phenylephrine, by up to 3-fold. The increased analyte signals observed in the chemical-vapor-assisted ESI process is related to the enhancement of the ionization efficiency in ESI. The method can be readily implemented to an existing ESI mass spectrometer at minimum cost for improving detection sensitivity. [ABSTRACT FROM AUTHOR]

In this study, the efficiency of a suspect screening strategy using liquid chromatography-high resolution mass spectrometry (LC-HRMS) without the prior purchase of reference standards was systematically optimized and evaluated for assessing the exposure of rarely investigated pesticides and their transformation products (TPs) in 76 surface water samples. Water-soluble and readily ionizable (electrospray ionization) substances, 185 in total, were selected from a list of all insecticides and fungicides registered in Switzerland and their major TPs. Initially, a solid phase extraction-LC-HRMS method was established using 45 known, persistent, and high sales volume pesticides. Seventy percent of these target substances had limit of quantitation (LOQ) < 5 ng L-1. This compound set was then used to develop and optimize a HRMS suspect screening method using only the exact mass as a priori information. Thresholds for blank subtraction, peak area, peak shape, signal-to-noise, and isotopic pattern were applied to automatically filter the initially picked peaks. The success rate was 70%; false negatives mainly resulted from low intense peaks. The optimized approach was applied to the remaining 140 substances. Nineteen additional substances were detected in environmental samples, two TPs for the first time in the environment. Sixteen substances were confirmed with reference standards purchased subsequently, while three TP standards could be obtained from industry or other laboratories. Overall, this screening approach was fast and very successful and can easily be expanded to other micropollutant classes for which reference standards are not readily accessible such as TPs of household chemicals. [ABSTRACT FROM AUTHOR]

A new analytical setup allowing the simultaneous analysis of precursors, intermediates and products in Maillard reaction model systems was developed to investigate the formation and mitigation kinetics of acrylamide. It was based on high-performance liquid chromatography combined with electrospray ionization triple quadrupole tandem mass spectrometry (HPLC- MS/MS). Chromatography and mass spectrometry conditions were optimized to permit simultaneous monitoring of compounds relevant to acrylamide, such as asparagine, glucose, glycine, N-(1- deoxy-D-fructos-1-yl)-glycine (DFG), N-(1-deoxy-D-fructos-1-yl)- asparagine (DFA), and four reaction products between acrylamide and glycine. A fairly good separation was achieved on a Venusil MP-C18 column using an isocratic eluent of 0.5% formic acid in water at a flow rate of 0.4 mL/min. Triple quadrupole mass spectrometry in multiple reaction monitoring mode (MRM) was applied to obtain good sensitivity. All of the ten key reaction compounds were separated and determined in a single run within 15 min. High correlation coefficients (r > 0.99) of the ten analytes were obtained in their respective linear ranges. The method was capable to accurately quantify the ten key compounds in model systems during heating. The results showed that acrylamide formed in Maillard reaction could be reduced by reacting directly with glycine, and acrylamide concentration constantly decreased by about 60% within the 180 min heating at 150°C when glycine was equal to asparagine and glucose in molecular concentration in model systems. This method helps achieve reduction in both time and labor of analysis of a large number of samples. [ABSTRACT FROM AUTHOR]

Despite the increasing biological interests on glycoconjugates, the synthetic mechanism of oligosaccharides has not yet been revealed except for the enzymes involved. To clarify the synthetic events that occur inside cells, spatiotemporal analysis of fluorescently tagged glycosphingolipids was carried out. Transformation of the incorporated lactosylceramide analogue carrying 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl group (BODIPY fluorophore) was analyzed using nanoLC-fluorescence detection-nanoelectrospray ionization-mass spectrometry. A complex process of glycan synthesis and degradation was our primary observation. [ABSTRACT FROM AUTHOR]

The gel-based proteomic analysis of plasma membranes from rat liver and chemically induced, malignant hepatocellular carcinoma Morris hepatoma 7777 was systematically optimized to yield the maximum number of proteins containing transmembrane domains (TMDs). Incorporation of plasma membrane proteins into a polyacrylamide "tube gel" followed by in-gel digestion of "tube gel" pieces significantly improved detection by electrospray ionization-liquid chromatography-tandem mass spectrometry. Removal of less hydrophobic proteins by washing isolated plasma membranes with 0.1 M sodium carbonate enables detection of a higher number of hydrophobic proteins containing TMDs in both tissues. Subsequent treatment of plasma membranes by a proteolytic enzyme (trypsin) causes the loss of some of the proteins that are detected after washing with sodium carbonate, but it enables the detection of other hydrophobic proteins containing TMDs. Introduction of mass spectrometers with higher sensitivity, higher mass resolution and mass accuracy, and a faster scan rate significantly improved detection of membrane proteins, but the improved sample preparation is still useful and enables detection of additional hydrophobic proteins. Proteolytic predigestion of plasma membranes enables detection of additional hydrophobic proteins and better sequence coverage of TMD-containing proteins in plasma membranes from both tissues. [ABSTRACT FROM AUTHOR]

Mass spectrometry-based platforms have gained increasing success in discovery of ligands bound to therapeutic targets as drug candidates. We established both a nanoelectrospray ionization mass spectrometry (nanoESI-MS) assay and an ultrafiltration liquid chromatography/mass spectrometry (LC/MS) assay to identify new ligands for New Delhi metallo-β-lactamase 1 (NDM-1), responsible for worldwide antibiotic resistance. To alleviate nonspecific binding of hydrophobic compounds and eliminate false positives typically encountered in the indirect LC/MS-based assay, we introduced a blocking protein in the control, which remarkably enhances the selectivity and accuracy of the indirect approach. Side-by-side comparison of the two MS-based approaches for the first time further reveals unique advantages of the indirect approach, including better reproducibility and tolerance of interference. Moreover, the success of fishing out a potent ligand from a mixture of small-molecule fragments demonstrates great potential of the indirect LC/MS-based approach for constructing a robust screening platform against combinatorial libraries or natural product extracts. More importantly, by combining the results of MS-based analyses, enzymatic activity assay, competition experiments, and structural simulation, we discovered a new compound as a promising drug candidate targeting NDM-1. [ABSTRACT FROM AUTHOR]

Here, we present the application of a cross-platform approach, combining rapid direct infusion high-resolution/accurate mass electrospray ionization Fourier transform ion-cyclotron mass spectrometry (ESI-FTICRMS) with in-depth data-dependent LC-MS² and LC-MS³ analysis for lipid profiling. The analytical approach as well as the subsequent data handling is described. The method was applied to human synovial fluid samples from osteo- and rheumatoid arthritis patients. Multivariate statistical analysis revealed esterified oxylipids as molecular features in a subset of the patient samples. Employing LC-MS² and LC-MS³ analysis of these species, we were able to clarify the hypothesized lipid structures initially based on the accurate mass measurements performed on the ESI-FTICRMS platform. LC-MS³ analysis of intact esterified oxy-lipids and LC-MS² analysis of the hydrolysis products allowed for the detection of positional isomers. The approach led to the structural elucidation of hydroxylated docosapentaenoic acid-containing diacyl-phosphatidylcholine type phospholipids in human synovial fluid. [ABSTRACT FROM AUTHOR]

The common derivatization method of primary amino groups by 2,4,6-trinitrobenzene sulfonate (TNBS) was applied to the detection of free adducts of advanced glycation end-products (AGEs), in combination with liquid chromatography-tandem mass spectrometry-multiple reaction monitoring (LC-MS/MS-MRM). The proposed TNBS-MS method provided a surprisingly significant improvement of the detection of AGE-free adducts (e.g., by a factor of >1000 for methylglyoxal-derived hydroimidazolone) with a detection limit of 1.0 nM (10 fmol/injection volume), which was due to the high ionization efficiency of the derived trinitrophenol moiety and its hydrophobicity. With the aid of stable-isotope-labeled internal standard (MG-H1-d3), the convenient TNBS method that allowed the derivation of AGE-free adducts bearing amino groups under mild reaction conditions (30 mM, pH 8.5, 30 min, 30 °C) also permitted successive detection and quantification of five typical AGE-free adducts at nanomolar levels in rat plasma (50 μL) with high reproducibility (2-9% of RSD) and recovery (93-113%), using LC-MS/MS-MRM. [ABSTRACT FROM AUTHOR]

Because routine preparation of glycan samples involves multiple reaction and cleaning steps at which sample loss occurs, glycan analysis is typically performed using large tissue samples. This type of analysis yields no detailed molecular spatial information and requires special care to maintain proper storage and shipping conditions. We describe here a new glycan sample preparation protocol using minimized sample preparation steps and optimized procedures. Tissue sections and spotted samples first undergo on-surface enzymatic digestion to release N-glycans. The released glycans are then reduced and permethylated prior to online purification and LC-electrospray ionization (ESI)-MS analysis. The efficiency of this protocol was initially evaluated using model glycoproteins and human blood serum (HBS) spotted on glass or Teflon slides. The new protocol permitted the detection of permethylated N-glycans derived from 10 ng RNase B. On the other hand, 66 N-glycans were identified when injecting the equivalent of permethylated glycans derived from a 0.1-μL aliquot of HBS. On-tissue enzymatic digestion of nude mouse brain tissue permitted the detection of 43 N-glycans. The relative peak areas of these 43 glycans were comparable to those from a C57BL/6 mouse reported by the Consortium for Functional Glycomics (CFG). However, the sample size analyzed in the protocol described here was substantially smaller than for the routine method (submicrogram vs mg). The on-tissue N-glycan profiling method permits high sensitivity and reproducibility and can be widely applied to assess the spatial distribution of glycans associated with tissue sections, and may be correlated with immunoflourescence imaging when adjacent tissue sections are analyzed. [ABSTRACT FROM AUTHOR]

Antibody-drug conjugates (ADCs), an increasingly important therapeutic modality for targeted cancer treatment, have been studied using many analytical methods. A class of ADCs that utilize the reduced interchain disulfide cysteine residues for drug attachment has attracted particular interest in drug development. One challenge in analytical characterization of this class of ADCs is that the intact mass information of the ADC molecule is not attainable using conventional reversed-phase liquid chromatography-mass spectrometry methods. In this paper, we report a mass spectrometry (MS) method engaging enzymatic digestion, nanoelectrospray ionization (nano-ESI), and native MS to achieve direct determination of the intact mass and, furthermore, to calculate the average drug-to-antibody ratio (DAR) of the cysteine-linked ADCs. The novel aspects of this method lie in the application of a nano-ESI technique and, more significantly, the utilization of limited enzymatic digestion with a cysteine protease as compared to the recently published method by Valliere-Douglass et al.(1) In summary, this novel native nano-ESI MS method in combination with limited enzymatic digestion provides a sensitive method for direct DAR determination and possesses great potential in studying low-abundance ADC analytes such as those from animal or human in vivo investigations. [ABSTRACT FROM AUTHOR]

The mechanism-based inactivation of cytochrome P450 2B4 (CYP2B4) by 9-ethynylphenanthrene (9EP) has been investigated. The partition ratio and kinact are 0.2 and 0.25 min-1, respectively. Intriguingly, the inactivation exhibits sigmoidal kinetics with a Hill coefficient of 2.5 and an S50 of 4.5 μM indicative of homotropic cooperativity. Enzyme inactivation led to an increase in mass of the apo-CYP2B4 by 218 Da as determined by electrospray ionization liquid chromatography and mass spectrometry, consistent with covalent protein modification. The modified CYP2B4 was purified to homogeneity and its structure determined by X-ray crystallography. The structure showed that 9EP is covalently attached to Oγ of Thr 302 via an ester bond, which is consistent with the increased mass of the protein. The presence of the bulky phenanthrenyl ring resulted in inward rotations of Phe 297 and Phe 206, leading to a compact active site. Thus, binding of another molecule of 9EP in the active site is prohibited. However, results from the quenching of 9EP fluorescence by unmodified or 9EP-modified CYP2B4 revealed at least two binding sites with distinct affinities, with the low-affinity site being the catalytic site and the high-affinity site on the protein periphery. Computer-aided docking and molecular dynamics simulations with one or two ligands bound revealed that the high-affinity site is situated at the entrance of a substrate access channel surrounded by the F' helix, β1-β2 loop, and β4 loop and functions as an allosteric site to enhance the efficiency of activation of the acetylenic group of 9EP and subsequent covalent modification of Thr 302. [ABSTRACT FROM AUTHOR]

High-throughput multiplexed proteomics of small- volume biospecimens will generate new opportunities in theranostics. Achieving parallel top-down and bottom-up mass spectrometry analyses of target proteins using a unified apparatus will improve proteome characterization. We have developed a novel silicon-based microfluidic device, multinozzle emitter array chip (MEA chip), as a new platform for small-volume proteomics using liquid chromatography-nanoelectrospray ionization mass spectrometry (LC-nanoESl- MS). We demonstrate parallel, on-chip, and online LC-MS analysis of hemoglobin and its tryptic digests directly from microliters of blood, achieving a detection limit of less than 5 red blood cells. Our MEA chip will enable clinical proteomics of small-volume samples. [ABSTRACT FROM AUTHOR]

Human serum glycan profiling with mass spectrometry (MS) has been employed to study several disease conditions and is demonstrating promise in, for example, clinical biomarker discovery. However, the low glycan ionization efficiency and the large dynamic range of glycan concentrations in human sera can hinder comprehensive profiling. In particular, large glycans are problematic because they are present at low concentrations and are prone to fragmentation. Here we show that, following liquid chromatographic separation on graphite columns, subambient pressure ionization with nanoelectrospray (SPIN)-MS can expand the serum glycome profile in comparison with the conventional atmospheric pressure electrospray ionization (ESI)-MS with a heated capillary inlet. Notably, the ions generated by the SPIN interface were observed at higher charge states for approximately half of the annotated glycans. Out of a total of 130 detected glycans, 34 were only detected with the SPIN-MS, resulting in improved coverage of glycan families as well as of glycans with larger numbers of labile monosaccharides. [ABSTRACT FROM AUTHOR]

Low molecular heparins (LMWHs) are structurally complex, heterogeneous, polydisperse, and highly negatively charged mixtures of polysaccharides. The direct characterization of LMWH is a major challenge for currently available analytical technologies. Electrospray ionization (ESI) liquid chromatography-mass spectrometry (LC-MS) is a powerful tool for the characterization complex biological samples in the fields of proteomics, metabolomics, and glycomics. LC-MS has been applied to the analysis of heparin oligosaccharides, separated by size exclusion, reversed phase ion-pairing chromatography, and chip-based amide hydrophilic interaction chromatography (HILIC). However, there have been limited applications of ESI-LC-MS for the direct characterization of intact LMWHs (top-down analysis) due to their structural complexity, low ionization efficiency, and sulfate loss. Here we present a simple and reliable HILIC-Fourier transform (FT)-ESI-MS platform to characterize and compare two currently marketed LMWH products using the top-down approach requiring no special sample preparation steps. This HILIC system relies on cross-linked diol rather than amide chemistry, affording highly resolved chromatographic separations using a relatively high percentage of acetonitrile in the mobile phase, resulting in stable and high efficiency ionization. Bioinformatics software (GlycReSoft 1.0) was used to automatically assign structures within 5-ppm mass accuracy. [ABSTRACT FROM AUTHOR]

We report the effects of supercharging reagents dimethyl sulphoxide (DMSO) and m-nitrobenzyl alcohol ( m-NBA) applied to untargeted peptide identification, with special emphasis on non-tryptic peptides. Peptides generated from a mixture of five standard proteins digested with trypsin, elastase, or pepsin were separated with nanoflow liquid chromatography using mobile phases modified with either 5 % DMSO or 0.1 % m-NBA. Eluting peptides were ionized by online electrospray and sequenced by both CID and ETD using data-dependent MS/MS. Statistically significant improvements in peptide identifications were observed with DMSO co-solvent. In order to understand this observation, we assessed the effects of supercharging reagents on the chromatographic separation and the electrospray quality. The increase in identifications was not due to supercharging, which was greater for the 0.1 % m-NBA co-solvent and not observed for the 5.0 % DMSO co-solvent. The improved MS/MS efficiency using the DMSO modified mobile phase appeared to result from charge state coalescence. [ABSTRACT FROM AUTHOR]

The use of narrow bore LC capillaries operated at altralow flow rates coupled with mass spectrometay provides a desirable convergence of figures of merit to support high- performance LC-MS/MS analysis. This configuration provides a viable means to achieve in-depth protein sequence coverage while maintaining a high rate of data production. Here we explore potential performance improvements afforded by use of 25 μm x 100 cm columns fabricated with 5 μdiameter reversed phase particles and integrated electrospray emitter tips. These columns achieve a separation peak capacity of ≈750 m a 600-mm gradient, with average chromatographic peak widths of less than 1 cain. At room temperature, a pressure drop of only es1500 psi is sufficient to maintain an effluent flow rate of ⩽10 nL/min. Using mouse embryonic stem cells as a model for complex mammalian proteomes, we reproducibly identify over 4000 proteins across duplicate 600 mm LC-MS/MS analyses. [ABSTRACT FROM AUTHOR]

Epsilon toxin (ETX) is one of the most lethal toxins produced by Clostridium species and is considered as a potential bioterrorist weapon. Here, we present a rapid mass spectrometry-based method for ETX quantification in complex matrixes. As a prerequisite, naturally occurring prototoxin and toxin species were first structurally characterized by top-down and bottom-up experiments, to identify the most pertinent peptides for quantification. Following selective ETX immunoextraction and trypsin digestion, two proteotypic peptides shared by all the toxin forms were separated by ultraperformance liquid chromatography (UPLC) and monitored by ESI-MS (electrospray ionization-mass spectrometry) operating in the multiple reaction monitoring mode (MEM) with collision-induced dissociation. Thorough protocol optimization, i.e., a 15 mm immunocapture, a 2 h enzymatic digestion, and an UPLC-MS/MS detection, allowed the whole quantification process including the calibration curve to be performed in less than 4 h, without compromising assay robustness and sensitivity. The assay sensitivity in milk and serum was estimated at S ng.mL-1 for ETX, making this approach complementary to enzyme linked immunosorbent assay (ELISA) techniques. [ABSTRACT FROM AUTHOR]

Enhanced charging, or supercharging, of analytes in electrospray ionization mass spectrometry (ESI MS) facilitates high resolution MS by reducing an ion mass-tocharge (m/z) ratio, increasing tandem mass spectrometry (MS/MS) efficiency. ESI MS supercharging is usually achieved by adding a supercharging reagent to the electrospray solution. Addition of these supercharging reagents to the mobile phase in liquid chromatography (LC)-MS/MS increases the average charge of enzymatically derived peptides and improves peptide and protein identification in large-scale bottom-up proteomics applications but disrupts chromatographic separation. Here, we demonstrate the average charge state of selected peptides and proteins increases by introducing the supercharging reagents directly into the ESI Taylor cone (in-spray supercharging) using a dual-sprayer ESI microchip. The results are comparable to those obtained by the addition of supercharging reagents directly into the analyte solution or LC mobile phase. Therefore, supercharging reaction can be accomplished on a time-scale of ion liberation from a droplet in the ESI ion source. [ABSTRACT FROM AUTHOR]

We demonstrate the use of capillary zone electrophoresis with an electrokinetically pumped sheath-flow electrospray interface for the analysis of a tryptic digest of a sample of intermediate protein complexity, the secreted protein fraction of Mycobacterium marinum. For electrophoretic analysis, 11 fractions were generated from the sample using reverse-phase liquid chromatography; each fraction was analyzed by CZE-ESI-MS/MS, and 334 peptides corresponding to 140 proteins were identified in 165 min of mass spectrometer time at 95% confidence (FDR < 0.15%). In comparison, 388 peptides corresponding to 134 proteins were identified in 180 min of mass spectrometer time by triplicate UPLC-ESI-MS/MS analyses, each using 250 ng of the unfractionated peptide mixture, at 95% confidence (FDR < 0.15%). Overall, 62% of peptides identified in CZE-ESI-MS/MS and 67% in UPLC-ESI-MS/MS were unique. CZE-ESI-MS/MS favored basic and hydrophilic peptides with low molecular masses. Combining the two data sets increased the number of unique peptides by 53%. Our approach identified more than twice as many proteins as the previous record for capillary electrophoresis proteome analysis. CE-ESI-MS/MS is a useful tool for the analysis of proteome samples of intermediate complexity. [ABSTRACT FROM AUTHOR]