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Synthetic biology seeks to reprogram microbial cells for efficient production of value-added compounds from low-cost renewable substrates. A great challenge of chemicals biosynthesis is the competition between cell metabolism and target product synthesis for limited cellular resource. Dynamic regulation provides an effective strategy for fine-tuning metabolic flux to maximize chemicals production. In this work, we created a tunable growth phase-dependent autonomous bifunctional genetic switch (GABS) by coupling growth phase responsive promoters and degrons to dynamically redirect the carbon flux for metabolic state switching from cell growth mode to production mode, and achieved high-level GABA production from low-value glycerol in Corynebacterium glutamicum. A ribosome binding sites (RBS)-library-based pathway optimization strategy was firstly developed to reconstruct and optimize the glycerol utilization pathway in C. glutamicum, and the resulting strain CgGly2 displayed excellent glycerol utilization ability. Then, the initial GABA-producing strain was constructed by deleting the GABA degradation pathway and introducing an exogenous GABA synthetic pathway, which led to 5.26 g/L of GABA production from glycerol. In order to resolve the conflicts of carbon flux between cell growth and GABA production, we used the GABS to reconstruct the GABA synthetic metabolic network, in which the competitive modules of GABA biosynthesis, including the tricarboxylic acid (TCA) cycle module and the arginine biosynthesis module, were dynamically down-regulated while the synthetic modules were dynamically up-regulated after sufficient biomass accumulation. Finally, the resulting strain G7-1 accumulated 45.6 g/L of GABA with a yield of 0.4 g/g glycerol, which was the highest titer of GABA ever reported from low-value glycerol. Therefore, these results provide a promising technology to dynamically balance the metabolic flux for the efficient production of other high value-added chemicals from a low-value substrate in C. glutamicum.

Native top-down proteomics allows for both proteoform identification and high-order structure characteri-zation for cellular protein complexes. Unfortunately, tandem MS-based fragmentation efficiencies for such targets are low due to an increase in analyte ion mass and the low ion charge states that characterize native MS data. Multiple fragmenta-tion methods can be integrated in order to increase protein complex sequence coverage, but this typically requires use of specialized hardware and software. Free-radical initiated peptide sequencing (FRIPS) enables access to charge-remote and electron-based fragmentation channels within the context of conventional CID experiments. Here, we optimize FRIPS la-belling for native top-down sequencing experiments. Our labelling approach is able to access intact complexes with TEMPO-based FRIPS reagents without significant protein denaturation or assembly disruption. By combining CID and FRIPS datasets, we observed sequence coverage improvements as large as 50% for protein complexes ranging from 36 kDa to 106 kDa. Fragment ion production in these experiments was increased by as much as 102%. In general, our results indicate that TEMPO-based FRIPS reagents have the potential to dramatically increase sequence coverage obtained in na-tive top-down experiments.

Despite recent advances in glycomics, glycan characterization still remains an analytical challenge. Accordingly, numerous glycan-tagging reagents with different chemistries were developed, including those involving acid-base chemistry and/or free radical chemistry. Acid-base chemistry excels at dissociating glycans into their constituent components in a systematic and predictable manner to generate cleavages at glycosidic bonds. Glycans are also highly susceptible to depolymerization by free radical processes, which is supported by results observed from electron-activated dissociation techniques. Therefore, the free radical activated glycan sequencing (FRAGS) reagent was developed so as to possess the characteristics of both acid-base and free radical chemistry, thus generating information-rich glycosidic bond and cross-ring cleavages. Alternatively, the free radical processes can be induced via photodissociation of the specific carbon-iodine bond which gives birth to similar fragmentation patterns as the FRAGS reagent. Furthermore, the methylated-FRAGS (Me-FRAGS) reagent was developed to eliminate glycan rearrangements by way of a fixed charged as opposed to a labile proton, which would otherwise yield additional, yet unpredictable, fragmentations including internal residue losses or multiple external residue losses. Lastly, to further enhance glycan enrichment and characterization, solid-support FRAGS was developed.

The gaseous 2,6-didehydropyridinium cation and its derivatives transfer a proton to reagents for which the reaction for their singlet ground states is too endothermic to be observed. These reaction...

Chemically enhanced primary treatment (CEPT) with ferrate(VI), a multifunctional treatment agent, was investigated for the treatment of municipal wastewater in a laboratory-scale study. The treatment performance was evaluated at different ferrate(VI) doses (0.0-9.0 mg/L as Fe) and pH (6.0 and 7.5). The optimal removals of total suspended solids (TSS) (52%), total chemical oxygen demand (COD) (34%), and total phosphorus (47%) were achieved at the highest ferrate(VI) dose (9.0 mg/L as Fe) and the weakly alkaline condition (pH 7.5). The pollutant abatements principally ascribed to the formation of large-sized aggregate and ensuing sedimentation fell within the reported ranges of CEPT with traditional coagulants. However, different from conventional CEPT, ferrate(VI) appreciably removed recalcitrant dissolved organic phosphorus (49%) and simultaneously inactivated total coliform (3.30 log removal) and Escherichia coli (3.67 log removal) at 9.0 mg/L Fe(VI) and pH 7.5. The CEPT with ferrate(VI) offers an innovative alternative for improving municipal wastewater treatment. PRACTITIONER POINTS: Ferrate(VI) represents a promising agent for chemically enhanced primary treatment (CEPT) of municipal wastewater. CEPT with ferrate(VI) can effectively alleviate TSS, total COD, and total P via the formation of large-sized aggregates and ensuing sedimentation. Ferrate(VI) can substantially remove recalcitrant dissolved organic phosphorus in municipal wastewater. Different from other CEPT coagulants, ferrate(VI) can appreciably inactivate bacterial indicators during CEPT. Higher ferrate(VI) dose and weakly alkaline pH favor the performance of ferrate(VI) CEPT.

The inherent structural complexity and diversity of glycans pose a major analytical challenge to their structural analysis. Radical chemistry has gained considerable momentum in the field of mass spectrometric biomolecule analysis, including proteomics, glycomics, and lipidomics. Herein, seven isomeric disaccharides and two isomeric tetrasaccharides with subtle structural differences are distinguished rapidly and accurately via one-step radical-induced dissociation. The free-radical-activated glycan-sequencing reagent (FRAGS) selectively conjugates to the unique reducing terminus of glycans in which a localized nascent free radical is generated upon collisional activation and simultaneously induces glycan fragmentation. Higher-energy collisional dissociation (HCD) and collision-induced dissociation (CID) are employed to provide complementary structural information for the identification and discrimination of glycan isomers by providing different fragmentation pathways to generate informative, structurally significant product ions. Furthermore, multiple-stage tandem mass spectrometry (MS(3) CID) provides supplementary and valuable structural information through the generation of characteristic parent-structure-dependent fragment ions.

Metabolic engineering seeks to rewire the metabolic network of cells for the efficient production of value-added compounds from renewable substrates. However, it remains challenging to evaluate and identify strains with the desired phenotype from the vast rational or random mutagenesis library. One effective approach to resolve this bottleneck is to design an efficient high-throughput screening (HTS) method to rapidly detect and analyze target candidates. L-cysteine is an important sulfur-containing amino acid and has been widely used in agriculture, pharmaceuticals, cosmetics, and food additive industries. However, HTS methods that enable monitoring of L-cysteine levels and screening of the enzyme variants and strains to confer superior L-cysteine biosynthesis remain unavailable, greatly limiting the development of efficient microbial cell factories for L-cysteine production at the industrial scale. Here, we took advantage of the L-cysteine-responsive transcriptional regulator CcdR to develop a genetically encoded biosensor for engineering and screening the L-cysteine overproducer. The in vivo L-cysteine-responsive assays and in vitro electrophoretic mobility shift assay (EMSA) and DNase I footprint analysis indicated that CcdR is a transcriptional activator that specifically interacts with L-cysteine and binds to its regulatory region to induce the expression of target genes. To improve the response performance of the L-cysteine biosensor, multilevel optimization strategies were performed, including regulator engineering by semi-rational design and systematic optimization of the genetic elements by modulating the promoter and RBS combination. As a result, the dynamic range and sensitivity of the biosensor were significantly improved. Using this the excellent L-cysteine biosensor, a HTS platform was established by coupling with fluorescence-activated cell sorting (FACS) and was successfully applied to achieve direct evolution of the key enzyme in the L-cysteine biosynthetic pathway to increase its catalytic performance and to screen the high L-cysteine-producing strains from the random mutagenesis library. These results presented a paradigm of design and optimization of biosensors to dynamically detect metabolite concentrations and provided a promising tool enabling HTS and metabolic regulation to construct L-cysteine hyperproducing strains to satisfy industrial demand.

Soybean is a worldwide crop that offers valuable proteins, fatty acids, and phytonutrients to humans but is always damaged by insect pests or pathogens. Plants have captured sophisticated defense mechanisms in resisting the attack of insects and pathogens. How to protect soybean in an environment- or human-friendly way or how to develop plant-based pest control is a hotpot. Herbivore-induced plant volatiles that are released by multiple plant species have been assessed in multi-systems against various insects, of which (E)-β-ocimene has been reported to show anti-insect function in a variety of plants, including soybean. However, the responsible gene in soybean is unknown, and its mechanism of synthesis and anti-insect properties lacks comprehensive assessment. In this study, (E)-β-ocimene was confirmed to be induced by Spodoptera litura treatment. A plastidic localized monoterpene synthase gene, designated as GmOCS, was identified to be responsible for the biosynthesis of (E)-β-ocimene through genome-wide gene family screening and in vitro and in vivo assays. Results from transgenic soybean and tobacco confirmed that (E)-β-ocimene catalyzed by GmOCS had pivotal roles in repelling a S. litura attack. This study advances the understanding of (E)-β-ocimene synthesis and its function in crops, as well as provides a good candidate for further anti-insect soybean improvement.

The gaseous 2,6-didehydropyridinium cation and its derivatives transfer a proton to reagents for which the reaction for their singlet ground states is too endothermic to be observed. These reactions occur from the lowest-energy excited triplet states, which has not been observed (or reported) for other

RATIONALE The biggest obstacle in the rational conversion of biomass into aromatic chemicals is the identification of unknown compounds in lignin degradation mixtures that are highly complex. As opposed to lignin degradation products with β-O-4 linkages, very little is known about the mass spectrometric analysis of lignin degradation products with α-O-4 linkages. METHODS Lignin model compounds with an α-O-4 and another linkage, as well as lignin model compounds with only β-O-4 linkages, were ionized by attachment of lithium or sodium cations under positive-ion mode electrospray ionization (ESI) or by deprotonation in negative-ion mode ESI in a linear quadrupole ion trap mass spectrometer. The ions were subjected to collision-activated dissociation in multiple-stage tandem mass spectrometry experiments to characterize their fragmentation patterns. RESULTS All studied compounds formed abundant sodium and lithium cation adducts in positive-ion mode ESI with no fragmentation. Model compounds with β-O-4 linkages displayed stable [M - H]- ions in negative-ion mode ESI whereas compounds with α-O-4 linkages only showed fragment ions. CAD of the lithiated α-O-4 compounds provided more structural information than CAD of sodiated compounds. However, both sodiated and lithiated compounds with α-O-4 linkages showed losses of monomer units at the MS2 stage, which is useful for sequencing of lignins with this type of linkage. CONCLUSIONS An ionization and sequencing method has been developed for lignin model compounds with α-O-4 linkages that spontaneously fragment upon ionization via (-)ESI.

2,4,6-Tridehydropyridinium cation (7) undergoes three consecutive atom or atom group abstractions from reagent molecules in the gas phase. By placing a π-electron-donating hydroxyl group between two radical sites, their reactivity can be quenched by enhancing their through-space coupling via a favorable resonance structure. Indeed, 3-hydroxy-2,4,6-tridehydropyridinium cation (8) abstracts only one atom or group of atoms from reagents. On the other hand, an electron-withdrawing cyano group between two of the radical sites (9) destabilizes the analogous resonance structure and diminishes through-space coupling between the radical sites, resulting in abstraction of three atoms, just like 7. However, the cyano-substituent also increases acidity to the point that 9 reacts pre-dominantly via proton transfer instead of undergoing radical reactions. Therefore, acidic triradicals may undergo nonradical, barrierless proton transfer reactions faster than radical reactions, which are usually accompanied by barriers. Examination of the analogous cyano-substituted mono-and biradicals revealed behavior similar to that of the corresponding unsubstituted species, with the exception of substantially greater reactivities due to their greater (calculated) vertical electron affinities. Finally, the 3-cyano-2,6-didehydropyridinium cation with a singlet ground state (S-T splitting: -11.9 kcal mol-1) was found to react exclusively from the lowest-energy triplet state by fast proton transfer reactions.

We report herein a gas-phase reactivity study on a para-benzyne cation and its three cyano-substituted, isomeric derivatives performed using a dual-linear quadrupole ion trap mass spectrometer. All four biradicals were found to undergo primary and secondary radical reactions analogous to those observed for the related monoradicals, indicating the presence of two reactive radical sites. The reactivity of all biradicals is substantially lower than that of the related monoradicals, as expected based on the singlet ground states of the biradicals. The cyano-substituted biradicals show substantially greater reactivity than the analogous unsubstituted biradical. The greater reactivity is rationalized by the substantially greater (calculated) electron affinity of the radical sites of the cyano-substituted biradicals, which results in stabilization of their transition states through polar effects. This finding is in contrast to the long-standing thinking that the magnitude of the singlet-triplet splitting controls the reactivity of para-benzynes.

Seven synthesized G-lignin oligomer model compounds (ranging in size from dimers to an octamer) with 5-5 and/or β-O-4 linkages, and three synthesized S-lignin model compounds (a dimer, trimer, and tetramer) with β-O-4 linkages, were evaporated and deprotonated using negative-ion mode ESI in a linear quadrupole ion trap/Fourier transform ion cyclotron resonance mass spectrometer. The collision-activated dissociation (CAD) fragmentation patterns (obtained in MS2 and MS3 experiments, respectively) for the negative ions were studied to develop a procedure for sequencing unknown lignin oligomers. On the basis of the observed fragmentation patterns, the measured elemental compositions of the most abundant fragment ions, and quantum chemical calculations, the most important reaction pathways and likely mechanisms were delineated. Many of these reactions occur via charge-remote fragmentation mechanisms. Deprotonated compounds with only β-O-4 linkages, or both 5-5 and β-O-4 linkages, showed major 1,2-eliminations ...

Corynebacterium glutamicum is generally regarded as a safe microorganism, and widely used in the large-scale production of various amino acids and organic acids, such as L-glutamate, L-lysine and succinic acid. During the process of industrial fermentation, C. glutamicum is usually exposed to varying environmental stresses, such as variations in pH, salinity, temperature, and osmolality. Among them, pH fluctuations are regarded as one of the most frequent environmental stresses in microbial fermentation. In this review, we summarize the current knowledge of pH homeostasis mechanisms adopted by C. glutamicum for coping with low acidic pH and high alkaline pH stresses. Facing with low pH environments, C. glutamicum develops a variety of strategies to maintain intracellular pH homeostasis, such as lowering intracellular reactive oxygen species, the improvement of potassium transport, the regulation of mycothiol-related pathways, as well as the repression of sulfur assimilation. While during alkaline pH stresses, the Mrp-type Na+/H+ antiporters are shown to play a dominant role in conferring C. glutamicum cells resistance to alkaline pH. Furthermore, we also discuss the general strategies and prospects on metabolic engineering of C. glutamicum to improve alkaline or acid resistance.

By combing the merits of solid supports and free radical activated glycan sequencing (FRAGS) reagents, we develop a multi-functional solid-supported free radical probe (SS-FRAGS), which enables glycan enrichment and characterization. SS-FRAGS comprises a solid support, free radical precursor, disulfide bond, pyridyl, and hydrazine moieties. Thio-activated resin and magnetic nanoparticles (MNPs) are chosen as the solid support to selectively capture free glycans via the hydrazine moiety, allowing for their enrichment and isolation. The disulfide bond acts as a temporary covalent linkage between the solid support and the captured glycan, allowing the release of glycans via the cleavage of the disulfide bond by dithiothreitol. The basic pyridyl functional group provides a site for the formation of a fixed charge, enabling detection by mass spectrometry and avoiding glycan rearrangement during collisional activation. The free radical precursor generates a nascent free radical upon collisional activation and thus simultaneously induces systematic and predictable fragmentation for glycan structure elucidation. A radical-driven glycan deconstruction diagram (R-DECON) is developed to visually summarize the MS(2) results and thus allow for the assembly of the glycan skeleton, making the differentiation of isobaric glycan isomers unambiguous. For application to a real-world sample, we demonstrate the efficacy of the SS-FRAGS by analyzing glycan structures enzymatically cleaved from RNase-B.

To rationally design less cytotoxic synthetic drugs based on biologically active para-benzyne intermediates, it is essential to obtain a better understanding on the reactivity of para-benzynes. However, little is still known about their fundamental chemical properties, which requires examination of pure species in simple solvents instead of biological matrices. While fundamental solution studies are limited, the solvent-free reactivity of protonated para-benzynes has been examined previously in the gas phase by using mass spectrometry. In order to facilitate the comparison of results obtained in the gas phase and in solution, we attempted to compare the reactivity of a neutral para-benzyne analog toward dimethyldisulfide in solution to that of its protonated form in the gas phase. The neutral para-benzyne analog, 5,8-didehydroquinoline, was successfully generated via photolysis of an enediyne precursor in dimethyldisulfide/methanol solution and the reaction products were analyzed using a dual linear quadrupole ion trap tandem mass spectrometrometer. However, examination of of the reactions of the same para-benzyne in its protonated form in the gas phase in the mass spectrometer proved to be challenging. Therefore, an analogous, isomeric para-benzyne cation, the 5,8-didehydroisoquinolinium cation, was instead generated in the gas phase from the 5-iodo-8-nitroisoquinoline precursor and its reactions with dimethyldisulfide were examined. This protonated para-benzyne reacted with dimethyl disulfide in the gas phase in the same manner as its neutral isomer in solution; both predominantly abstracted a thiomethyl group by each of the two radical sites. The structures of these products were verified by tandem mass spectrometry experiments based on collision-activated dissociation and using an authentic model compound. These findings suggest that the reactivity of the studied para-benzynes toward dimethyldisulfide is similar in spite of their different protonation states and the different phases in which the reactions were carried out.

Detailed glycan structural characterization is frequently achieved by collisionally activated dissociation (CAD) based sequential tandem mass spectrometry (MS(n)) analysis of permethylated glycans. However, it is challenging to implement MS(n) (n > 2) during online glycan separation, and this has limited its application to analysis of complex glycan mixtures from biological samples. Further, permethylation can reduce liquid chromatographic (LC) resolution of isomeric glycans. Here, we studied the electronic excitation dissociation (EED) fragmentation behavior of native glycans with a reducing-end fixed charge tag and identified key spectral features that are useful for topology and linkage determination. We also developed a de novo glycan sequencing software that showed remarkable accuracy in glycan topology elucidation based on the EED spectra of fixed charge-derivatized glycans. The ability to obtain glycan structural details at the MS(2) level, without permethylation, via a combination of fixed charge derivatization, EED, and de novo spectral interpretation, makes the present approach a promising tool for comprehensive and rapid characterization of glycan mixtures.

Leaf petiole angle (LPA) is an important plant architectural trait that affects canopy coverage, photosynthetic efficiency, and ultimately productivity in many legume crops. However, the genetic basis underlying this trait remains unclear. Here, we report the identification, isolation, and functional characterization of

Wireless network technology’s widely used brings the web’s users huge convenience and flexibility, and also brings great challenges for the security of network.This paper introduces the MAC address authentication, Wired Equivalent Privacy (WEP), Wi-Fi Protected Access (WPA), WiFi Protected Access 2 (WPA2) and 802.1X and so on, giving priority to analysing their pros and cons. A new model will be used to cope with the security issues of WIFI based on it.

We designed and synthesized a methylated free radical activated glycan sequencing reagent (Me-FRAGS) for eliminating mass spectrometric glycan rearrangement., Mass spectrometric glycan rearrangement is problematic because it provides misleading structural information. Here we report on a new reagent, a methylated free radical activated glycan sequencing reagent (Me-FRAGS), which combines a free radical precursor with a methylated pyridine moiety that can be coupled to the reducing terminus of glycans. The collisional activation of Me-FRAGS-derivatized glycans generates a nascent free radical that concurrently induces abundant glycosidic bond and cross-ring cleavage without the need for subsequent activation. The product ions resulting from glycan rearrangement, including internal residue loss and multiple external residue losses, are precluded. Glycan structures can be easily assembled and visualized using a radical driven glycan deconstruction diagram (R-DECON diagram). The presence and location of N-acetylated saccharide units and branch sites can be identified from the characteristic dissociation patterns observed only at these locations. The mechanisms of dissociation are investigated and discussed. This Me-FRAGS based mass spectrometric approach creates a new blueprint for glycan structure analysis.

The influence of the type of fermentation method on phenolics, antioxidant capacity, and volatiles in blackberry wine was studied. Dry blackberry wines made by traditional fermentation (TF) and carbonic maceration fermentation (CMF) were analyzed for total polyphenols, flavanols, flavonoids, anthocyanins, proanthocyanidin, and antioxidant capacity. High-performance liquid chromatography was used for analysis of nonflavonoid phenolics (gallic, benzoic, salicylic, syringic, caffeic, coumaric, and ferulic) and flavonoids (catechin, quercetin, and rutin). Volatiles were detected by gas chromatography-mass spectrometry. The results showed that CMF fermentation afforded higher antioxidant activity and phenolic content, especially individual polyphenolics. The total level of phenolics in the CMF wine was substantially higher than in traditional wines: 2953 mg of gallic acid equivalents (GAE)/L for CMF wine vs. 1647 mg of GAE/L for traditional wine. A total of 53 kinds of volatile compounds were detected. Of thes...

Wines made from 3 spine grape (Vitis davidii Foex) genotypes—Junzi 1# (JZ 1#), Junzi 2# (JZ 2#), and Liantang (LT)—and Cherokee rose (Rosa laevigata Michx., CR) were evaluated for their phenolics composition and antioxidant activities by several assays, including 2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS scavenging capacity, cupric ion reducing antioxidant capacity, and metal-chelating capacity). The results showed that CR wine had higher contents of total phenolics, total flavonoids, and oligomeric proanthocyanidins than the 3 spine grape genotype wines and Cabernet Sauvignon (CS) wine. Among the 3 varieties of spine grape wines, JZ 1# had higher contents than the 2 other genotypes. Moreover, the total monomeric anthocyanins in JZ 1# was about 4.5-fold higher than those in CS wine and CR wine. A significance analysis demonstrated that the antioxidant capacity (as measured by the ABTS scavenging capacity and cupric ion reducing antioxidant capacity) of CR wine was significantly higher than that of the control and spine grape wines. With respect to metal-chelating capacity, the CR wine was the strongest amongst all the wine samples tested, followed by JZ 1#. This indicates that CR and JZ 1# wines can potentially be considered as wild fruit wines with abundant phenolic compounds and antioxidant activities. Practical Application: Spine grape (Vitis davidii Foex) and Cherokee rose (Rosa laevigata Michx.) are 2 species of important wild plants in South China. However, they have not yet been effectively developed and utilized. Recently, some researchers attempted to convert their juice to wine. We anticipate that this research will provide sufficient experimental evidence for their good antioxidant activity and potential for further development and utilization.

While atmospheric pressure ionization methodologies have revolutionized the mass spectrometric analysis of nonvolatile analytes, limitations native to the chemistry of these methodologies hinder or entirely inhibit the analysis of certain analytes, specifically, many nonpolar compounds. Examination of various analytes, including asphaltene and lignin model compounds as well as saturated hydrocarbons, demonstrates that atmospheric pressure chemical ionization (APCI) using CS2 as the reagent produces an abundant and stable molecular ion (M+•) for all model compounds studied, with the exception of completely saturated aliphatic hydrocarbons and the two amino acids tested, arginine and phenylalanine. This reagent substantially broadens the applicability of mass spectrometry to nonvolatile nonpolar analytes and also facilitates the examination of radical cation chemistry by mass spectrometry. Copyright © 2011 John Wiley & Sons, Ltd.

Distinct from template-directed biosynthesis of nucleic acids and proteins, the enzymatic synthesis of heterogeneous polysaccharides is a complex process that is difficult to study using common analytical tools. Therefore, the mode of action and processivity of those enzymes are largely unknown. Dermatan sulfate epimerase 1 (DS-epi1) is the predominant enzyme during the formation of iduronic acid residues in the glycosaminoglycan dermatan sulfate. Using recombinant DS-epi1 as a model enzyme, we describe a tandem mass spectrometry-based method to study the mode of action of polysaccharide processing enzymes. The enzyme action on the substrate was monitored by hydrogen–deuterium exchange mass spectrometry and the sequence information was then fed into mathematical models with two different assumptions of the mode of action for the enzyme: processive reducing end to non-reducing end, and processive non-reducing end to reducing end. Model data was scored by correlation to experimental data and it was found that DS-epi1 attacks its substrate on a random position, followed by a processive mode of modification towards the non-reducing end and that the substrate affinity of the enzyme is negatively affected by each additional epimerization event. It could also be shown that the smallest active substrate was the reducing end uronic acid in a tetrasaccharide and that octasaccharides and longer oligosaccharides were optimal substrates. The method of using tandem mass spectrometry to generate sequence information of the complex enzymatic products in combination with in silico modeling can be potentially applied to study the mode of action of other enzymes involved in polysaccharide biosynthesis.

We investigate the mechanism of disulfide bond cleavage in gaseous peptide and protein ions initiated by a covalently-attached regiospecific acetyl radical using mass spectrometry (MS). Highly selective S–S bond cleavages with some minor C–S bond cleavages are observed by a single step of collisional activation. We show that even multiple disulfide bonds in intact bovine insulin are fragmented in the MS2 stage, releasing the A- and B-chains with a high yield, which has been challenging to achieve by other ion activation methods. Yet, regardless of the previous reaction mechanism studies, it has remained unclear why (1) disulfide bond cleavage is preferred to peptide backbone fragmentation, and why (2) the S–S bond that requires the higher activation energy conjectured in previously suggested mechanisms is more prone to be cleaved than the C–S bond by hydrogen-deficient radicals. To probe the mechanism of these processes, model peptides possessing deuterated β-carbon(s) at the disulfide bond are employed. It is suggested that the favored pathway of S–S bond cleavage is triggered by direct acetyl radical attack at sulfur with concomitant cleavage of the S–S bond (S_H2). The activation energy for this process is substantially lower by ~9–10 kcal mol^(−1) than those of peptide backbone cleavage processes determined by density functional quantum chemical calculations. Minor reaction pathways are initiated by hydrogen abstraction from the α-carbon or the β-carbon of a disulfide, followed by β-cleavages yielding C–S or S–S bond scissions. The current mechanistic findings should be generally applicable to other radical-driven disulfide bond cleavages with different radical species such as the benzyl and methyl pyridyl radicals.

A fast-pyrolysis probe/tandem mass spectrometer combination was utilized to determine the initial fast-pyrolysis products for four different selectively (13)C-labeled cellobiose molecules. Several products are shown to result entirely from fragmentation of the reducing end of cellobiose, leaving the nonreducing end intact in these products. These findings are in disagreement with mechanisms proposed previously. Quantum chemical calculations were used to identify feasible low-energy pathways for several products. These results provide insights into the mechanisms of fast pyrolysis of cellulose.

Free radical-initiated peptide sequencing (FRIPS) mass spectrometry derives advantage from the introduction of highly selective low-energy dissociation pathways in target peptides. An acetyl radical, formed at the peptide N-terminus via collisional activation and subsequent dissociation of a covalently attached radical precursor, abstracts a hydrogen atom from diverse sites on the peptide, yielding sequence information through backbone cleavage as well as side-chain loss. Unique free-radical-initiated dissociation pathways observed at serine and threonine residues lead to cleavage of the neighboring N-terminal C_α–C or N–C_α bond rather than the typical Cα–C bond cleavage observed with other amino acids. These reactions were investigated by FRIPS of model peptides of the form AARAAAXAA, where X is the amino acid of interest. In combination with density functional theory (DFT) calculations, the experiments indicate the strong influence of hydrogen bonding at serine or threonine on the observed free radical chemistry. Hydrogen bonding of the side-chain hydroxyl group with a backbone carbonyl oxygen aligns the singly occupied π orbital on the β-carbon and the N–C_α bond, leading to low-barrier β-cleavage of the N–C_α bond. Interaction with the N-terminal carbonyl favors a hydrogen-atom transfer process to yield stable c and z• ions, whereas C-terminal interaction leads to effective cleavage of the C_α–C bond through rapid loss of isocyanic acid. Dissociation of the C_α–C bond may also occur via water loss followed by β-cleavage from a nitrogen-centered radical. These competitive dissociation pathways from a single residue illustrate the sensitivity of gas-phase free radical chemistry to subtle factors such as hydrogen bonding that affect the potential energy surface for these low-barrier processes.

A novel immunoassay of methyltestosterone (MT) in serum is developed. We synthesized the antigen of MT (AgMT) by covalently bonding it to BSA and raised its antibody (AbMT) from rabbit. AbMT was conjugated to poly-N-isopropylacrylamide (p-NIPAAm) to form AbMT bound thermally reversible hydrogel (p-NIPAAm-AbMT). A competitive immunoassay method based on the competition of fluorescein isothiocyanate (FITC) labeled MT antigen (FITC-AgMT) and MT with limited amounts of p-NIPAAm-AbMT was established. The separation in the assay process was achieved by precipitation of the immuno-complex above its critical solution temperature. The detection limit of MT is 50 ng/ml. The recoveries of MT in human serum are above 93%.

A group of soluble polymer-supported chiral tartrate ligands was prepared by liquid-phase synthesis with ligand diversity strategy. Moderate to excellent chemical yields and enantiomeric excesses were obtained by using soluble polymer-supported tartrate ester in asymmetric oxidation of prochiral sulfides using Ti(O- i -Pr) 4 /cumyl hydroperoxide, and the workup was greatly simplified. The influence of substituent in chiral tartrate ligands on the enantioselectivities of the reaction was disclosed.

A mass spectrometric method utilizing gas-phase ion-molecule reactions of 1-butanethiol and di-tert-butyl peroxide has been developed for the differentiation of primary, secondary and tertiary hydroxyl functionalities in protonated analytes in a FT-ICR mass spectrometer.

Recent studies have shown that the reactivity of the 4-dehydropyridinium cation significantly differs from the reactivities of its isomers toward tetrahydrofuran. While only hydrogen atom abstraction was observed for the 2- and 3-dehydropyridinium cations, nonradical reactions were observed for the 4-isomer. In order to learn more about these reactions, the gas-phase reactivities of the 4-dehydropyridinium cation and several of its derivatives toward tetrahydrofuran were investigated in a Fourier transform ion electron resonance mass spectrometer. Both radical and nonradical reactions were observed for most of these positively charged radicals. The major parameter determining whether nonradical reactions occur was found to be the electron affinity of the radicals--only those with relatively high electron affinities underwent nonradical reactions. The reactivities of the monoradicals are also affected by hydrogen bonding and steric effects.

Saturated and unsaturated, linear, branched, and cyclic hydrocarbons, as well as polyaromatic and heteroaromatic hydrocarbons, were successfully ionized by atmospheric pressure chemical ionization (APCI) using small hydrocarbons as reagents in a linear quadrupole ion trap (LQIT) mass spectrometer. Pentane was proved to be the best reagent among the hydrocarbon reagents studied. This ionization method generated different types of abundant ions (i.e., [M + H](+), M(+•), [M - H](+) and [M - 2H](+ •)), with little or no fragmentation. The radical cations can be differentiated from the even-electron ions by using dimethyl disulfide, thus facilitating molecular weight (MW) determination. While some steroids and lignin monomer model compounds, such as androsterone and 4-hydroxy-3-methoxybenzaldehyde, also formed abundant M(+•) and [M + H](+) ions, this was not true for all of them. Analysis of two known mixtures as well as a base oil sample demonstrated that each component of the known mixtures could be observed and that a correct MW distribution was obtained for the base oil. The feasibility of using this ionization method on the chromatographic time scale was demonstrated by using high-performance liquid chromatography (HPLC) with hexane as the mobile phase (and APCI reagent) to separate an artificial mixture prior to mass spectrometric analysis.

While atmospheric pressure ionization methodologies have revolutionized the mass spectrometric analysis of nonvolatile analytes, limitations native to the chemistry of these methodologies hinder or entirely inhibit the analysis of certain analytes, specifically, many nonpolar compounds. Examination of various analytes, including asphaltene and lignin model compounds as well as saturated hydrocarbons, demonstrates that atmospheric pressure chemical ionization (APCI) using CS(2) as the reagent produces an abundant and stable molecular ion (M(+•)) for all model compounds studied, with the exception of completely saturated aliphatic hydrocarbons and the two amino acids tested, arginine and phenylalanine. This reagent substantially broadens the applicability of mass spectrometry to nonvolatile nonpolar analytes and also facilitates the examination of radical cation chemistry by mass spectrometry.

Laser-induced acoustic desorption (LIAD) was successfully coupled to a conventional atmospheric pressure chemical ionization (APCI) source in a linear quadrupole ion trap mass spectrometer (LQIT). Model compounds representing a wide variety of different types, including basic nitrogen and oxygen compounds, aromatic and aliphatic compounds, as well as unsaturated and saturated hydrocarbons, were tested separately and as a mixture. These model compounds were successfully evaporated into the gas phase by using LIAD and then ionized by using APCI with different reagents. Four APCI reagent systems were tested: the traditionally used mixture of methanol and water, neat benzene, neat carbon disulfide, and nitrogen gas (no liquid reagent). The mixture of methanol and water produced primarily protonated molecules, as expected. However, only the most basic compounds yielded ions under these conditions. In sharp contrast, using APCI with either neat benzene or neat carbon disulfide as the reagent resulted in the ionization of all the analytes studied to predominantly yield stable molecular ions. Benzene yielded a larger fraction of protonated molecules than carbon disulfide, which is a disadvantage. A similar amount of fragmentation was observed for these reagents. When the experiment was performed without a liquid reagent(nitrogen gas was the reagent), more fragmentation was observed. Analysis of a known mixture as well as a petroleum cut was also carried out. In summary, the new experiment presented here allows the evaporation of thermally labile compounds, both polar and nonpolar, without dissociation or aggregation, and their ionization to form stable molecular ions.

This paper presents a real-time sound source localization system based on switched capacitor networks and logic circuits. The system employs three microphones at different locations. Low pass filter circuits, noise reduction circuits and valid speech detecting circuits are implemented with dpASP(dynamically programmable Analog Signal Processor). A data latching array is constructed to record the order of the wavefront of sound signal. A decoder circuit designed output the sound source direction according to the state of the data latching array. The proposed system can output sound source direction in real-time with low power.

A group of soluble polymer-supported chiral tartrate ligands was prepared by liquid-phase synthesis with ligand diversity strategy. Moderate to excellent chemical yields and enantiomeric excesses were obtained by using soluble polymer-supported tartrate ester in asymmetric oxidation of prochiral sulfides using Ti(O- i -Pr) 4 /cumyl hydroperoxide, and the workup was greatly simplified. The influence of substituent in chiral tartrate ligands on the enantioselectivities of the reaction was disclosed.