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Sulfite, a widely used food additive, is subject to regulated labeling. The extraction of sulfite as the stable hydroxymethylsulfonate (HMS) form and its quantitative analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been recognized for their good sensitivity, selectivity, and versatility across various food materials. This study aimed to develop a cost-effective and simpler method for sulfite quantitation, while maintaining the superior sensitivity and selectivity of mass spectrometry (MS). To achieve this, we introduced paper spray ionization (PSI), an ambient desorption ionization technique that could achieve the direct measurement of analytes without employing separation. We also employed a novel internal standard (IS) structurally similar to the analyte, replacing the more expensive isotopically labeled IS. Although the PSI-MS/MS method developed in this study exhibited slightly lower analytical performance compared to the conventional LC-MS/MS, it remained effective for sulfite analysis in dried fruits. [ABSTRACT FROM AUTHOR]

Paper spray ionization mass spectrometry (PSI MS) has emerged as a notable method for the rapid analysis of biological samples. However, the typical cellulose-based paper tip is incompatible with protein detection due to the strong interaction between cellulose hydroxyl groups and proteins. In this study, we utilized a commercially available polyolefin-based synthetic paper, Teslin®, as an alternative PSI substrate for simple protein analysis. We have named this method "droplet PSI" MS, as the aqueous protein solution droplet retains its shape on the Teslin® paper tip. For droplet PSI, no further chemical pretreatment was necessary for the Teslin® substrate; the only required preparation was shaping the Teslin® paper into a triangular tip. In droplet PSI MS, protein ion signals were instantly detected from a protein solution droplet upon applying a spray solvent in situ along with high voltage (HV). When compared with conventional PSI MS, our method demonstrated superior sensitivity. The droplet PSI MS utilizing Teslin® also showcased flexibility in real-time observation of protein alterations induced by an acid additive. Additionally, the effects of spray solvent composition and the application method were discussed. [ABSTRACT FROM AUTHOR]

Nanospray desorption electrospray ionization (nano-DESI) is one of the ambient desorption ionization methods for mass spectrometry (MS), and it utilizes a steady-state liquid junction formed between two microcapillaries to directly extract analytes from sample surfaces with minimal sample damage. In this study, we employed nano-DESI MS to perform a metabolite fingerprinting analysis directly from a Hypericum leaf surface. Moreover, we investigated whether changes in metabolite fingerprints with time can be related to metabolite distribution according to depth. From a raw Hypericum leaf, the mass spectral fingerprints of key metabolites, including flavonoids and prenylated phloroglucinols, were successfully obtained using ethanol as a nano-DESI solvent, and the changes in their intensities were observed with time via full mass scan experiments. In addition, the differential extraction patterns of the obtained mass spectral fingerprints were clearly visualized over time through selected ion monitoring and pseudo-selected reaction monitoring experiments. To examine the correlation between the time-dependent changes in the metabolite fingerprints and depth-wise metabolite distribution, we performed a nano-DESI MS analysis against leaves whose surface layers were removed multiple times by forming polymeric gum Arabic films on their surfaces, followed by detaching. The preliminary results showed that the changes in the metabolite fingerprints according to the number of peelings showed a similar pattern with those obtained from the raw leaves over time. [ABSTRACT FROM AUTHOR]

Colloidal silver laser desorption/ionization (LDI) mass spectrometry (MS) was employed to directly profile and image epicuticular wax metabolites on a variety of different surfaces of Arabidopsis thaliana leaves and flowers. Major cuticular wax compounds, such as very long-chain fatty acids, alcohols, alkanes, and ketones, were successfully detected as silver adduct ions. The surface metabolites of different flower organs (carpels, petals, and sepals) were profiled for the first time at a spatial resolution of ~100 [micro]m. In addition, mass spectral profiles and images were collected from wild type and a mutant strain, which carried alleles that affect the surface constituents of this organism. One of these mutant alleles (cer2-2) is in a gene whose biochemical functionality is still unclear, although its effect on normal epicuticular wax deposition was the characteristic that led to its original identification. Variations of wax products between different spatial locations for wild type and for a mutant strain were investigated by normalizing the ion intensities to a reference peak ([[[sup.107]Ag + [sup.109]Ag].sup.+]). The spatially resolved surface metabolite profiling data of this mutant has provided new insights into the complexity of epicuticular wax deposition at the cellular-resolution scale. This MS-based metabolite imaging technology has the potential to provide valuable data for dissecting metabolism in multicellular organism at the level of single cells.

Due to a high background in the low-mass region, conventional MALDI is not as useful for detecting small molecules (molecular masses < 500 Da) as it is for large ones. Also, spatial inhomogeneity that is inherent to crystalline matrixes can degrade resolution in imaging mass spectrometry (IMS). In this study, colloidal graphite was investigated as an alternative matrix for laser desorption/ionization (GALDI) in IMS. We demonstrate its advantages over conventional MALDI in the detection of small molecules such as organic acids, flavonoids, and oligosaccharides. GALDI provides good sensitivity for such small molecules. The detection limit of fatty acids and flavonoids in the negative-ion mode are in the low-femtomole range. Molecules were detected directly and identified by comparing the MS and MS/MS spectra with those of standards. Various fruits were chosen to evaluate the practical utility of GALDI since many types of small molecules are present in them. Distribution of these small molecules in the fruit was investigated by using IMS and IMS/MS.

Graphite-assisted laser desorption/ionization (GALDI) mass spectrometry (MS) was investigated for analysis of cerebrosides in a complex total brain lipid extract. Conventional MALDI MS and GALDI MS were compared regarding lipid analysis by using high-vacuum (HV, < [10.sup.-6] Torr) LDI time-of-flight mass spectrometry and intermediate-pressure (IP, 0.17 Torr) linear ion trap mass spectrometry. Cerebrosides were not detected or detected with low sensitivity in MALDI MS because of other dominant phospholipids. By using GALDI, cerebrosides were detected as intense mass peaks without prior separation from other lipid species while mass peaks corresponding to phosphatidylcholines (PCs) were weak. The signal increase for cerebrosides and the signal decrease for PCs in GALDI MS were more significant in HV than in IP. M[S.sup.n] experiments of precursor ions corresponding to cerebrosides and PCs in brain lipid extract were performed to identify the detected species and distinguish isobaric ions. Twenty-two cerebroside species were detected by GALDI whereas eight cerebroside species were detected by MALDI. Sulfatides in brain lipid extract were also easily detected by GALDI MS in the negative ion mode. By forming a colloidal graphite thin film on rat brain tissue, direct lipid profiling by imaging mass spectrometry (IMS) was performed. Chemically selective images for cerebrosides and sulfatides were successfully obtained. Imaging tandem mass spectrometry (IMS/MS) was performed to generate images of specific product ions from isobaric species.

Gangliosides are amphiphilic, acidic glycosphingolipids possessing one or more sialic acid residues and several isobaric structural isomers with different abundances and bioactivities. Therefore, the distinction between these isomers is crucial for their proper profiling. Although liquid chromatography–mass spectrometry has been successfully employed for this purpose, the distinction process can still be improved, particularly regarding liquid chromatography. Recently, a reversed‐phase liquid chromatography method that could separate disialoganglioside isomers was reported; however, the distinction of trisialoganglioside isomers using reversed‐phase liquid chromatography has not been demonstrated. Here, we investigated the practicality of a reversed‐phase liquid chromatography with an octadecylsilane column for separating polysialoganglioside isomers and successfully achieved the isomer separation of disialogangliosides and trisialogangliosides for the first time. We also confirmed several crucial factors in the mobile‐phase composition, which affect the differential retention and mass spectral response of the isomers. First, an organic modifier, acetonitrile, exhibited superior selectivity against polysialogangliosides over methanol. Second, ammonium bicarbonate was the best ammonium salt additive among those tested, in terms of the separation efficiency and mass spectral response. Third, as the ammonium salt concentration increased, the negative electrospray ionization response was extensively suppressed, and the retention of gangliosides increased. [ABSTRACT FROM AUTHOR]

Paper spray chemical ionization (PSCI) combined with mass spectrometry has been proposed as a sensitive method for the analysis of nonpolar aromatic compounds; however, the mechanism behind PSCI is not well understood. In the present study, the evidence for the occurrence of corona discharge is provided and its mechanism is proposed. Photographs taken with a highly sensitive camera evidently demonstrate the occurrence of corona discharge at the end of the triangular shape tip when a nonpolar solvent such as hexane was used at an applied potential of 6–7 kV. Nevertheless, corona discharge was not observed in the presence of a polar solvent. The occurrence of the corona discharge was attributed to charge accumulation in the dielectric layer generated by the nonpolar solvent on the fibers of the paper tip. Specifically, corona discharge was generated at the tip end when the charge approached a critical threshold. In the presence of a polar solvent, however, the dielectric layer was not generated and, hence, corona discharge was not observed. Based on this information, three nonpolar solvents were selected and their sensitivity for analyzing the phenanthrene and maltene fractions of crude oil was evaluated. Chlorobenzene provided the highest signal abundance; therefore, it was suggested as the optimum solvent for PSCI. Notably, the fundamental understanding of corona discharge in PSCI acquired in this study provides a basis for further improvement of this technique by way of surface modification. [ABSTRACT FROM AUTHOR]

Reactive ionization with atmospheric pressure ionization (API) methods have been utilized for mass spectrometry (MS) to enhance ionization efficiency of an analyte, to recognize a specific functional group of an analyte, and to investigate reaction pathways or intermediates for a certain in situ reaction. Paper spray ionization (PSI) is one of the major API techniques for reactive ionization. In reactive PSI, one of the reactants is usually spotted onto a paper tip and the other reactant is delivered through a spraying solution. After in situ reaction, reaction products are transported to the end of the tip and ionized. In this study, we investigated reactions between isothiazolinones (ITs) and cysteamine (CA) by reactive PSI MS. An IT is a heterocyclic compound that has a conjugated ketone moiety and used as a biocide or a preservative. CA is a stable aminothiol that is often used as a bifunctional covalent modifier. Through reactive PSI MS of ITs with CA, we found that a covalent modification of an IT by CA was possible and sensitivities for ITs were improved compared to conventional PSI MS of ITs that did not employ CA. In addition, possible reaction pathways between ITs and CA during reactive PSI were suggested although we could not confirm a specific reaction pathway in the current study. [ABSTRACT FROM AUTHOR]

Citrullination is a post‐translational modification that converts arginine into citrulline through deimination. As citrullination is accompanied by only about 1 Da increase in mass, ion signals of a citrullinated peptide in a mass spectrum are always overlapped with the isotope peaks of its control (a noncitrullinated form). Therefore, off‐line, covalent labeling techniques on citrulline residues were usually employed to investigate citrullinated sites by mass spectrometry (MS). Recently, reactive desorption electrospray ionization (reactive DESI) MS was introduced as a tool for selective, in situ modification of target analytes. In reactive DESI MS, a chemical reagent that can readily react with a target analyte is sprayed onto a sample spot and resulting products are analyzed by MS. Here, we developed a reactive DESI MS method for determining the citrullinated sites in peptides by employing phenylglyoxal as a derivatization agent, and succeeded in selectively modifying a citrulline residue in a peptide. [ABSTRACT FROM AUTHOR]

Paper cone spray ionization ( PCSI), an extractive ambient ionization method that is specialized for raw solid material analysis, was applied to chemical fingerprinting analysis of raw medicinal herbs. Experimental parameters of PCSI mass spectrometry ( MS) were first optimized against Salvia miltiorrhiza Bunge (danshen). Our results showed that simple alkyl alcohols, such as methanol and 2-propanol, generally worked well in both polarities and could generate representative chemical fingerprints instantly for a given herbal sample. In addition, in situ extraction from untreated herbal slices during PCSI MS was found to be enough to generate characteristic chemical signatures of a given medicinal herb. After optimization, representative fingerprints of various medicinal herbs, including Sophora flavescens (kushen) and Paeonia lactiflora Pall (shaoyao), were successfully obtained by PCSI MS. In addition, morphologically similar herbs, Cynanchum auriculatum and Cynanchum wilfordii, could be differentiated from each other by their chemical signatures by PCSI MS. [ABSTRACT FROM AUTHOR]

We developed a modified paper spray ionization (PSI) platform, named as paper cone spray ionization (PCSI), for direct mass spectrometric analysis of raw solid samples. The major modification from conventional PSI was the geometry of the paper base: PCSI uses a triangular-pyramidal-shaped paper tip instead of a planar triangular-shaped one. This three-dimensional paper cone serves as a sample container, an in situ solid–liquid extraction chamber, an analyte transport channel, as well as an electrospray tip. Unlike conventional PSI mass spectrometry (MS), a weighing paper with relatively low permeability was found to be more suitable for PCSI MS than other papers. PCSI MS rapidly generated major chemical fingerprints from various solid materials including powdered tablets as well as raw and processed food materials without any sample pre-treatment. [ABSTRACT FROM AUTHOR]

RATIONALE: Quantifying polymers by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) with a conventional crystalline matrix generally suffers from poor sample-to-sample or shot-toshot reproducibility. An ionic-liquid matrix has been demonstrated to mitigate these reproducibility issues by providing a homogeneous sample surface, which is useful for quantifying polymers. In the present study, we evaluated the use of an ionic liquid matrix, i.e., 1-methylimidazolium α-cyano-4-hydroxycinnamate (1-MeIm-CHCA), to quantify polyhexamethylene guanidine (PHMG) samples that impose a critical health hazard when inhaled in the form of droplets. METHODS: MALDI-TOF mass spectra were acquired for PHMG oligomers using a variety of ionic-liquid matrices including 1-MeIm-CHCA. Calibration curves were constructed by plotting the sum of the PHMG oligomer peak areas versus PHMG sample concentration with a variety of peptide internal standards. RESULTS: Compared with the conventional crystalline matrix, the 1-MeIm-CHCA ionic-liquid matrix had much better reproducibility (lower standard deviations). Furthermore, by using an internal peptide standard, good linear calibration plots could be obtained over a range of PMHG concentrations of at least 4 orders of magnitude. CONCLUSIONS: This study successfully demonstrated that PHMG samples can be quantitatively characterized by MALDI-TOFMS with an ionic-liquid matrix and an internal standard. [ABSTRACT FROM AUTHOR]

Paper spray ionization (PSI) is an extractive ambient ionization technique for mass spectrometry (MS), whereby a triangular paper tip serves as the sampling base and the electrospray tip. During PSI, analytes are extracted and transported to the edge of the paper tip by the applied spraying solvent. Analytes can be purified from a sample matrix and separated from each other by this transportation process. In this study, we investigated and utilized the analyte transportation process of PSI for the in situ separation and analysis of lipid mixtures. We found that differential transport of phosphatidylcholine (PC) and triacylglycerol (TAG), the two most abundant lipid classes in animals, occurred during PSI. We also found that the order in which these lipids moved strongly depended on how the spraying solvent was applied to the paper base. The more polar PC moved faster than the less polar TAG during PSI, when a polar solvent was slowly fed into a paper tip, whereas TAG was transported faster than PC when excess solvent was applied to the tip at once. In addition, we achieved a complete separation and detection of PC and TAG by slowly supplying a nonpolar solvent to a PSI tip. [ABSTRACT FROM AUTHOR]

Many studies have analyzed nicotine metabolites in blood and urine to determine the toxicity caused by smoking, and assess exposure to cigarettes. Recently, hair and nails have been used as alternative samples for the evaluation of smoking, as not only do they reflect long-term exposure but they are also stable and easy to collect. Liquid-liquid or solid-phase extraction has mainly been used to detect nicotine metabolites in biological samples; however, these have disadvantages, such as the use of toxic organic solvents and complex pretreatments. In this study, a modified QuEChERS method was proposed for the first time to prepare samples for the detection of nicotine metabolite cotinine (COT) and trans-3′-hydroxycotinine (3-HCOT) in hair and nails. High-performance liquid chromatography–tandem mass spectrometry (LC–MS/MS) was used to analyze traces of nicotine metabolites. The established method was validated for selectivity, linearity, lower limit of quantitation, accuracy, precision and recovery. In comparison with conventional liquid-liquid extraction (LLE), the proposed method was more robust, and resulted in higher recoveries with favorable analytical sensitivity. Using this method, clinical samples from 26 Korean infants were successfully analyzed. This method is expected to be applicable in the routine analysis of nicotine metabolites for environmental and biological exposure monitoring. [ABSTRACT FROM AUTHOR]

Gangliosides, acidic glycosphingolipids with one or more sialic acid residues, are challenging to analyze with matrix-assisted laser desorption/ionization ( MALDI) mass spectrometry ( MS) because loss of sialic acid residues is significant during ionization process, especially in the positive-ion mode. Recently, we showed that 5-methoxysalicylic acid ( MSA) could be a matrix of choice for ganglioside analysis in the positive-ion mode. In this study, we investigated whether GD1a and GD1b, structural isomers with two sialic acid residues, are distinguishable by MALDI MS or tandem MS ( MS/ MS) with MSA and a proper salt additive. Our results showed that characteristic fragment ions of GD1a and GD1b were successfully observed by the positive-ion mode MALDI MS/ MS and these diagnostic ions were more reliably detected with increased intensities when an alkali earth metal additive such as CaCl2 was employed instead of an alkali metal additive such as NaCl. [ABSTRACT FROM AUTHOR]

Arginine residues in a protein can be enzymatically deiminated to citrulline residues, and this post-translational modification of proteins, called citrullination, has been known to be associated with autoimmune diseases such as rheumatoid arthritis. Since citrullination of an arginine residue causes a mass shift of only 1 Da, isotopic distribution patterns of citrullinated and its control (non-citrullinated) peptides overlap each other, and the resulting isotopic pattern is skewed with respect to that of the control peptide. Recently, Deforce and coworkers utilized the citrullination-induced skewed isotopic patterns for estimating the extent of citrullination ( J. Proteome Res. 2012, 11, 5245). However, electrospray ionization ( ESI), which Deforce and coworkers employed, is not fully suitable for this type of quantification because the number of potential charge sites on a citrullinated peptide is less than that on its control peptide and this leads to different ion abundances for a given charge state. In this study, we employed matrix-assisted laser desorption/ionization ( MALDI) instead of ESI for quantification of citrullination because the preference for singly charged ion formation by MALDI may overcome the aforementioned issue. Our results show that the isotopic patterns generated by MALDI time-of-flight ( TOF) mass spectrometry ( MS) reflect the citrullination contents better than those generated by ESI linear ion trap ( LIT) MS. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • First application of PSI-MS on photodegraded and degraded spilled oil samples. • PSI-MS is shown to be a suitable technique for spilled oil analysis. • Parameter derived from PSI-MS spectra to estimate the degree of degradation of oils. Abstract Paper spray ionization mass spectrometry (PSI-MS) was applied for the first time to study temporal change of photo-oxidized and weathered oils subjected to degradation. PSI is chosen in this study because it is an optimal ionization technique for the analysis of degraded oils with limited sample quantity and prone to salt and particulate contamination. With PSI-MS, quantitative analysis of oils can be successfully performed with as little as 2 μg of oil sample. In addition, oil solutions containing up to 0.05% sodium chloride were successfully analyzed with PSI-MS. In the PSI-MS spectra of photo-degraded oils, the relative abundance of compounds having double equivalence value (DBE) ≥ 5 increased but those with DBE < 5 decreased in number. The summed abundance ratio of compounds having DBE < 5 and DBE ≥ 5 showed a negative exponential correlation with the duration of UV exposure in laboratory experiments. The same trend was observed from spilled oils obtained from the environment. Therefore, this ratio serves as an effective means to estimate the degree of weathering in spilled oils. [ABSTRACT FROM AUTHOR]

Environmental pollutants (EPOLs), such as phthalates, volatile organic compounds, phenols, parabens, polycyclic aromatic hydrocarbons, pyrethroids, and environmental tobacco smoke, are highly heterogeneous compounds. Recently, attention has been drawn to the assessment of the combinatory effects of multiple EPs. To correlate multiple exposures with potential health implications, advanced comprehensive analytical methods covering multiclass EPOLs are essential. However, because of several technical problems associated with enzyme hydrolysis, simultaneous extraction, and multiresidue liquid chromatography–tandem mass spectrometry analysis, it is difficult to establish a comprehensive method covering a number of EPOLs in a single sample preparation and analytical run. We developed tandem hybrid hydrolysis, modified direct injection, and a comprehensive mobile phase to overcome these technical problems and established a comprehensive analytical method for simultaneous biomonitoring of multiclass EPOLs. Tandem hybrid hydrolysis using β-glucuronidase and consecutive acid hydrolysis allowed selective hydrolysis of glucuronide- and sulfate-conjugated metabolites without phthalate degradation. The comprehensive mobile phase composed of 0.01% acetic acid and acetonitrile enabled us to simultaneously analyze 86 EPOLs, with good chromatographic behavior and ionization efficiency. Modified direct injection allowed a small amount of sample and simultaneous urinary extraction. The method was validated and applied to 39 urine samples from 19 mother–newborn pairs for multiple exposure assessment. Results showed that BP-3, a general component in sunblock products, and monoethyl phthalate, a metabolite of diethyl phthalate, exhibit a clear positive correlation between mothers and newborns. Therefore, the developed method has potential as a novel analytical tool for long-term, large-scale, and data-rich human biomonitoring of EPOLs. • Comprehensive LC–ESI/MS/MS method for multiclass environmental pollutants. • Tandem hybrid hydrolysis and direct injection method without extraction. • Comprehensive mobile phase composition consisted of 0.01% acetic acid. • Analytical tool suitable for large-scale human biomonitoring. [ABSTRACT FROM AUTHOR]

Abstract: Enzymatic conversion of arginine to citrulline by peptidyl arginine deiminase is associated with peptide presentation and development of autoimmunity in rheumatoid arthritis. In order to facilitate identification of the citrullination site, citrulline residue was modified using 4-bromophenyl glyoxal, and 194Da mass increase and incorporation of the Br signature were confirmed by MALDI-TOF MS. Using this approach, we identified four and five citrullination sites of bovine serum albumin and bovine fibrinogen, respectively. MALDI-TOF/TOF MS was used to unambiguously identify two citrullination sites from bovine fibrinogen. [Copyright &y& Elsevier]

A genomic and spectroscopic signature-based search revealed a cycloaromatized enediyne, jejucarboside A ( 1 ), from a marine actinomycete strain. The structure of 1 was determined as a new cyclopenta[ a ]indene glycoside bearing carbonate functionality by nuclear magnetic resonance, high-resolution mass spectrometry (MS), MS/MS, infrared spectroscopy, and a modified Mosher's method. An iterative enediyne synthase pathway has been proposed for the putative biosynthesis of 1 by genomic analysis. Jejucarboside A exhibited cytotoxicity against the HCT116 colon carcinoma cells.

Paper spray ionization (PSI) is an extractive ambient ionization technique for mass spectrometry (MS), whereby a triangular paper tip serves as the sampling base and the electrospray tip. During PSI, analytes are extracted and transported to the edge of the paper tip by the applied spraying solvent. Analytes can be purified from a sample matrix and separated from each other by this transportation process. In this study, we investigated and utilized the analyte transportation process of PSI for the in situ separation and analysis of lipid mixtures. We found that differential transport of phosphatidylcholine (PC) and triacylglycerol (TAG), the two most abundant lipid classes in animals, occurred during PSI. We also found that the order in which these lipids moved strongly depended on how the spraying solvent was applied to the paper base. The more polar PC moved faster than the less polar TAG during PSI, when a polar solvent was slowly fed into a paper tip, whereas TAG was transported faster than PC when excess solvent was applied to the tip at once. In addition, we achieved a complete separation and detection of PC and TAG by slowly supplying a nonpolar solvent to a PSI tip.

Herein, gas-phase polycyclic aromatic hydrocarbons (PAHs) as nonpolar compounds were ionized to protonated molecular ions [M + H] + without radical cations and simultaneously analyzed using gas chromatography (GC)/electrospray ionization (ESI)-tandem mass spectrometry (MS/MS). The ionization profile, dissociation, and sensitivity were first investigated to understand the significant behavior of gas-phase PAHs under ESI. The formation of protonated molecular ions of PAHs was distinguished according to the analyte phase and ESI spray solvents. The protonated PAHs exhibited characteristic dissociations, such as H-loss, H 2 -loss, and acetylene-loss, via competition of internal energy. In addition, GC/ESI-MS/MS resulted in relatively lower concentration levels (better sensitivity) for the limits-of-detection (LODs) of PAHs than liquid chromatography (LC)/ESI-MS/MS, and it seems to result from the characteristic ionization mechanism of the gas-phase analyte under ESI. Furthermore, the LODs of gas-phase PAHs depended on molecular weight and proton affinity (PA). Consequently, we demonstrated the relationship among the analyte phases, sensitivities, and structural characteristics (molecular weight and PA) under ESI. The gas-phase PAHs provided enhanced protonation efficiency and sensitivity using GC/ESI-MS/MS, as their molecular weight and PA increased. Based on these results, we offered important information regarding the behavior of gas-phase analytes under ESI. Therefore, the present GC/ESI-MS/MS method has potential as an alternative method for simultaneous analysis of PAHs.

In this study, a hydrogen/deuterium (H/D) exchange method using gas chromatography-electrospray ionization/mass spectrometry (GC-ESI/MS) was first investigated as a novel tool for online H/D exchange of multitarget analytes. The GC and ESI source were combined with a homemade heated column transfer line. GC-ESI/MS-based H/D exchange occurs in an atmospheric pressure ion source as a result of reacting the gas-phase analyte eluted from GC with charged droplets of deuterium oxide infused as the ESI spray solvent. The consumption of the deuterated solvent at a flow rate of 2 μL min -1 was more economical than that in online H/D exchange methods reported to date. In-ESI-source H/D exchange by GC-ESI/MS was applied to 11 stimulants with secondary amino or hydroxyl groups. After H/D exchange, the spectra of the stimulants showed unexchanged, partially exchanged, and fully exchanged ions showing various degrees of exchange. The relative abundances corrected for naturally occurring isotopes of the fully exchanged ions of stimulants, except for etamivan, were in the range 24.3-85.5%. Methylephedrine and cyclazodone showed low H/D exchange efficiency under acidic, neutral, and basic spray solvent conditions and nonexchange for etamivan with an acidic phenolic OH group. The in-ESI-source H/D exchange efficiency by GC-ESI/MS was sufficient to determine the number of hydrogen by elucidation of fragmentation from the spectrum. Therefore, this online H/D exchange technique using GC-ESI/MS has potential as an alternative method for simultaneous H/D exchange of multitarget analytes.

Sensitivity is an important factor determining successful mass spectrometry (MS) analysis of metabolome, protein, drugs, and environmental samples. Currently, nanoelectrospray ionization (ESI) is widely used as a sensitive ionization method. However, application of nano-ESI is limited to polar molecules and there is no atmospheric pressure ionization technique developed that can be used for MS analysis of low- and nonpolar compounds with sensitivity that can match with nano-ESI. Herein, we propose paper spray chemical ionization (PSCI) as an ionization technique that can be used to analyze low- and nonpolar aromatic compounds with high sensitivity. PSCI is based on paper spray ionization utilizing corona discharge phenomenon. PSCI can sensitively and quantitatively detect down to picogram (or femtomole) levels of low- and nonpolar aromatic compounds.

Common saprophytic fungi are seldom present in Protea infructescences, which is strange given the abundance of mainly dead plant tissue in this moist protected environment. We hypothesized that the absence of common saprophytic fungi in Protea infructescences could be due to a special symbiosis where the presence of microbes producing antifungal compounds protect the infructescence. Using a culture based survey, employing selective media and in vitro antifungal assays, we isolated antibiotic producing actinomycetes from infructescences of Protea repens and P . neriifolia from two geographically separated areas. Isolates were grouped into three different morphological groups and appeared to be common in the Protea spp. examined in this study. The three groups were supported in 16S rRNA and multi-locus gene trees and were identified as potentially novel Streptomyces spp. All of the groups had antifungal activity in vitro . Streptomyces sp. Group 1 had inhibitory activity against all tested fungi and the active compound produced by this species was identified as fungichromin. Streptomyces spp. Groups 2 and 3 had lower inhibition against all tested fungi, while Group 3 showed limited inhibition against Candida albicans and Sporothrix isolates. The active compound for Group 2 was also identified as fungichromin even though its production level was much lower than Group 1. The antifungal activity of Group 3 was linked to actiphenol. The observed antifungal activity of the isolated actinomycetes could contribute to protection of the plant material against common saprophytic fungi, as fungichromin was also detected in extracts of the infructescence. The results of this study suggest that the antifungal Streptomyces spp. could play an important role in defining the microbial population associated with Protea infructescences.

Gene and protein expression changes observed with tumorigenesis are often interpreted independently of each other and out of context of biological networks. To address these limitations, this study examined several approaches to integrate transcriptomic and proteomic data with known protein-protein and signaling interactions in estrogen receptor positive (ER+) breast cancer tumors. An approach that built networks from differentially expressed proteins and identified among them networks enriched in differentially expressed genes yielded the greatest success. This method identified a set of genes and proteins linking pathways of cellular stress response, cancer metabolism, and tumor microenvironment. The proposed network underscores several biologically intriguing events not previously studied in the context of ER+ breast cancer, including the overexpression of p38 mitogen-activated protein kinase and the overexpression of poly(ADP-ribose) polymerase 1. A gene-based expression signature biomarker built from this network was significantly predictive of clinical relapse in multiple independent cohorts of ER+ breast cancer patients, even after correcting for standard clinicopathological variables. The results of this study demonstrate the utility and power of an integrated quantitative proteomic, transcriptomic, and network analysis approach to discover robust and clinically meaningful molecular changes in tumors.