Electrostatically regulated active site assembly governs reactivity in non-heme iron halogenases.

Non-heme iron halogenases (NHFe-Hals) catalyze the direct insertion of a chloride/bromide ion at an unactivated carbon position using a high-valent haloferryl intermediate. Despite more than a decade of structural and mechanistic characterization, how NHFe-Hals preferentially bind specific anions and substrates for C-H functionalization remains unknown. Herein, using lysine halogenating BesD and HalB enzymes as model systems, we demonstrate strong positive cooperativity between anion and substrate binding to the catalytic pocket.

Structural and Functional Characterization of Homoserine Transacetylase.

() lacking functional homoserine transacetylase (HTA) is compromised in methionine biosynthesis, protein synthesis, and in the activity of multiple essential -adenosyl-l-methionine-dependent enzymes. Additionally, deficient mutants are further disarmed by the toxic accumulation of lysine due to a redirection of the metabolic flux toward the lysine biosynthetic pathway. Studies with deletion mutants and crystallographic studies of the apoenzyme have, respectively, validated HTA as an essential enzyme and revealed a ligandable binding site.

Unconventional aliphatic fluorophores discovered as the luminescence origin in citric acid-urea carbon dots.

Carbon dots (CDs) are emerging as the material of choice in a range of applications due to their excellent photoluminescence properties, ease of preparation from inexpensive precursors, and low toxicity. However, the precise nature of the mechanism for the fluorescence is still under debate, and several molecular fluorophores have been reported. In this work, a new blue fluorophore, 5-oxopyrrolidine-3-carboxylic acid, was discovered in carbon dots synthesized from the most commonly used precursors: citric acid and urea.

Toward a rapid method for the study of biodiversity in cold environments: the characterization of psychrophilic yeasts by MALDI-TOF mass spectrometry.

To investigate the potential of matrix-assisted laser desorption ionization mass spectrometry (MALDI-TOF/MS) as a platform to support biodiversity and phylogenetic studies of psychrophilic yeasts in cold environments, the technique was employed to rapidly characterize and distinguish three psychrophilic yeasts (Rhodotorula mucilaginosa, Naganishia vishniacii, and Dioszegia cryoxerica) from three mesophilic counterparts (Saccharomyces cerevisiae Cry Havoc, S. cerevisiae California V Ale, and S. pastorianus).

Stereochemistry- and concentration-dependent effects of phosphatidylserine enrichment on platelet function.

Platelets are small (1-2μm in diameter), circulating anuclear cell fragments with important roles in hemostasis and thrombosis that provide an excellent platform for studying the role of membrane components in cellular communication. Platelets use several forms of communication including exocytosis of three distinct granule populations, formation of bioactive lipid mediators, and shape change (allowing for adhesion). This work explores the role of stereochemistry and concentration of exogenous phosphatidylserine (PS) on platelet exocytosis and adhesion.

Checkpoints for Preliminary Identification of Small Molecules found Enriched in Autophagosomes and Activated Mast Cell Secretions Analyzed by Comparative UPLC/MS.

We report the use of ultra high performance liquid chromatography (UPLC) coupled with acquisition of low- and high-collision energy mass spectra (MS) to explore small molecule compositions that are unique to either enriched-autophagosomes or secretions of chemically activated murine mast cells. Starting with thousands of features, each defined by a chromatographic retention time, values and ion intensities, manual examination of the extracted ion chromatograms (XIC) of chemometrically selected features was essential to eliminate false positives, occurring at rates of 33, 14 and 37% in samples of three biological systems. Forty-six percent of features that passed the XIC-based checkpoint, had IDs in compound databases used here.

Determination of agmatine using isotope dilution UPLC-tandem mass spectrometry: application to the characterization of the arginine decarboxylase pathway in Pseudomonas aeruginosa.

A method has been developed for the direct determination of agmatine in bacterial culture supernatants using isotope dilution ultra performance liquid chromatography (UPLC)-tandem mass spectrometry (UPLC-MS/MS). Agmatine determination in bacterial supernatants is comprised of spiking culture or isolate supernatants with a fixed concentration of uniformly labeled (13)C5,(15)N4-agmatine (synthesized by decarboxylation of uniformly labeled (13)C6,(15)N4-arginine using arginine decarboxylase from Pseudomonas aeruginosa) as an internal standard, followed by derivatization with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBDF) to improve the reversed-phase chromatographic retention characteristics of agmatine, as well as the selectivity and sensitivity of UPLC-MS/MS detection of this amine in complex biologically derived mixtures. Intrasample precisions for measurement of agmatine in culture supernatants average 4.

Analytical characterization of the role of phospholipids in platelet adhesion and secretion.

The cellular phospholipid membrane plays an important role in cell function and cell-cell communication, but its biocomplexity and dynamic nature presents a challenge for examining cellular uptake of phospholipids and the resultant effects on cell function. Platelets, small anuclear circulating cell bodies that influence a wide variety of physiological functions through their dynamic secretory and adhesion behavior, present an ideal platform for exploring the effects of exogenous phospholipids on membrane phospholipid content and cell function. In this work, a broad range of platelet functions are quantitatively assessed by leveraging a variety of analytical chemistry techniques, including ultraperformance liquid chromatography-tandem electrospray ionization mass spectrometry (UPLC-MS/MS), vasculature-mimicking microfluidic analysis, and single cell carbon-fiber microelectrode amperometry (CFMA).

The arginine decarboxylase pathways of host and pathogen interact to impact inflammatory pathways in the lung.

The arginine decarboxylase pathway, which converts arginine to agmatine, is present in both humans and most bacterial pathogens. In humans agmatine is a neurotransmitter with affinities towards α2-adrenoreceptors, serotonin receptors, and may inhibit nitric oxide synthase. In bacteria agmatine serves as a precursor to polyamine synthesis and was recently shown to enhance biofilm development in some strains of the respiratory pathogen Pseudomonas aeruginosa.

Site-specific labeling of proteins and peptides with trans-cyclooctene containing handles capable of tetrazine ligation.

There is a growing library of functionalized non-natural substrates for the enzyme protein farnesyltransferase (PFTase). PFTase covalently attaches these functionalized non-natural substrates to proteins ending in the sequence CAAX, where C is a cysteine that becomes alkylated, A represents an aliphatic amino acid, and X is Ser, Met, Ala, or Gln. Reported substrates include a variety of functionalities that allow modified proteins to undergo subsequent bioconjugation reactions.

Time- and concentration-dependent effects of exogenous serotonin and inflammatory cytokines on mast cell function.

Mast cells play a significant role in both the innate and adaptive immune response; however, the tissue-bound nature of mast cells presents an experimental roadblock to performing physiologically relevant mast cell experiments. In this work, a heterogeneous cell culture containing primary culture murine peritoneal mast cells (MPMCs) was studied to characterize the time-dependence of mast cell response to allergen stimulation and the time- and concentration-dependence of the ability of the heterogeneous MPMC culture to uptake and degranulate exogenous serotonin using high performance liquid chromatography (HPLC) coupled to an electrochemical detector. Additionally, because mast cells play a central role in asthma, MPMCs were exposed to CXCL10 and CCL5, two important asthma-related inflammatory cytokines that have recently been shown to induce mast cell degranulation.

On the potential of mass spectrometry-based metabolite profiling approaches to the study of biochemical adaptation in psychrophilic yeast.

To move beyond targeted approaches to the biochemical characterization of psychrophilic yeast and provide a more holistic understanding of the chemistry of physiological adaptation of psychrophiles at the molecular level, ultraperformance liquid chromatography combined with simultaneous acquisition of low- and high-collision energy mass spectra (UPLC/MS(e)) was employed for a preliminary comparative analysis of cell extracts of psychrophilic Antarctic yeasts Cryptococcus vishniacii CBS 10616 and Dioszegia cryoxerica CBS 10919 versus the mesophile Saccharomyces cerevisiae 'cry havoc'. A detailed workflow for providing high-confidence preliminary identifications of psychrophilic yeast-specific molecular features is presented. Preliminary identifications of psychrophile-specific features in C.

Isotope-dilution UPLC-MS/MS determination of cell-secreted bioactive lipids.

Secreted bioactive lipids play critical roles in cell-to-cell communication and have been implicated in inflammatory immune responses such as anaphylaxis, vasodilation, and bronchoconstriction. Analysis of secreted bioactive lipids can be challenging due to their relatively short lifetimes and structural diversity. Herein, a method has been developed using ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) to quantify five cell-secreted, structurally and functionally diverse bioactive lipids (PGD2, LTC4, LTD4, LTE4, PAF) that play roles in inflammation.

Oxy intermediates of homoprotocatechuate 2,3-dioxygenase: facile electron transfer between substrates.

Substrates homoprotocatechuate (HPCA) and O(2) bind to the Fe(II) of homoprotocatechuate 2,3-dioxygenase (FeHPCD) in adjacent coordination sites. Transfer of an electron(s) from HPCA to O(2) via the iron is proposed to activate the substrates for reaction with each other to initiate aromatic ring cleavage. Here, rapid-freeze-quench methods are used to trap and spectroscopically characterize intermediates in the reactions of the HPCA complexes of FeHPCD and the variant His200Asn (FeHPCD−HPCA and H200N−HPCA, respectively) with O(2).

Liquid chromatography/tandem mass spectrometry of glycolytic intermediates: deconvolution of coeluting structural isomers based on unique product ion ratios.

A method has been developed for rapid quantification of nine glycolytic intermediates using ultraperformance liquid chromatography/electrospray-tandem mass spectrometry (UPLC/ESI-MS/MS) to monitor the metabolism of glucose during microbial fermentation. Because comprehensive chromatographic separation is not required, analysis time is significantly less than traditional ion exchange liquid chromatography assays or enzymatic assays. Complete glycolytic intermediate analysis by LC/MS/MS can be achieved in less than 7 min per sample.

Profiling of organic acids during fermentation by ultraperformance liquid chromatography-tandem mass spectrometry.

A method has been developed for rapid quantification of organic acids using ultraperformance liquid chromatography/electrospray-tandem mass spectrometry (UPLC/ESI-MS-MS) to monitor the metabolism of 10 organic acids during microbial fermentation. Because comprehensive chromatographic separation is not required, analysis time is less than traditional ion chromatography assays, with complete organic acid analyses by UPLC/ESI-MS-MS being achieved in less than 3 min. Quantification is accomplished using nine isotopically labeled organic acids as internal standards.

Discovery of enzymatic activity using stable isotope metabolite labeling and liquid chromatography-mass spectrometry.

Stable isotope labeling of an intracellular chemical precursor or metabolite allows direct detection of downstream metabolites of that precursor, arising from novel enzymatic activity of interest, using metabolite profiling liquid chromatography-mass spectrometry. This approach allows the discrimination of downstream metabolites produced from novel enzymatic activity from the unlabeled form of the metabolite arising from native metabolic processes within the cell. Even for the case in which a given product of novel enzymatic activity is a transient, the novel enzymatic activity that produced it can be demonstrated to exist indirectly by identification of product-specific downstream metabolites.

Capillary electrophoresis for the investigation of prostate-specific antigen heterogeneity.

Prostate-specific antigen (PSA) is a single-chain glycoprotein that is used as a biomarker for prostate-related diseases. PSA has one known posttranslational modification, a sialylated diantennary N-linked oligosaccharide attached to the asparagine residue N45. In this study capillary electrophoresis (CE) was employed to separate the isoforms of seven commercially available free PSA samples, two of which were specialized: enzymatically active PSA and noncomplexing PSA.

Potential of fermentation profiling via rapid measurement of amino acid metabolism by liquid chromatography-tandem mass spectrometry.

Monitoring amino acid metabolism during fermentation has significant potential from the standpoint of strain selection, optimizing growth and production in host strains, and profiling microbial metabolism and growth state. A method has been developed based on rapid quantification of underivatized amino acids using liquid chromatography-electrospray tandem mass spectrometry (LC-MS-MS) to monitor the metabolism of 20 amino acids during microbial fermentation. The use of a teicoplanin-based chiral stationary phase coupled with electrospray tandem mass spectrometry allows complete amino acid analyses in less than 4 min.

Simultaneous identification of soyasaponins and isoflavones and quantification of soyasaponin Bb in soy products, using liquid chromatography/electrospray ionization-mass spectrometry.

A method has been developed for simultaneous identification of soyasaponins and soy isoflavones in soy products, based on liquid chromatography/electrospray ionization-mass spectrometry (LC/ESI-MS). Soy-based nutraceutical products were analyzed by LC/ESI-MS with detection of protonated and sodiated molecular ions, as well as characteristic fragment ions for these compounds. Soy isoflavones were characterized by a strong protonated molecular ion in addition to corresponding [aglycone + H](+) ions.

Separation and identification of organic acid-coenzyme A thioesters using liquid chromatography/electrospray ionization-mass spectrometry.

A method has been developed for the direct determination of coenzyme A (CoA) and organic acid-CoA thioesters in mixtures using directly combined liquid chromatography/electrospray ionization-mass spectrometry (LC/ESI-MS). Mixtures of CoA and organic acid-CoA thioesters were analyzed by LC/ESI-MS with detection of protonated molecular ions and characteristic fragment ions for each compound. The identities of the CoA-thioesters were established based on LC retention times and simultaneously recorded mass spectra.

Mass spectrometry: an emerging alternative to traditional methods for measurement of diagnostic proteins, peptides and amino acids.

Novel approaches to protein measurement based on mass spectrometry are being developed that challenge more traditional methods. This review summarizes the emergence of mass spectrometry as a tool for clinical protein, peptide, and amino acid determination. Specific applications of mass spectrometry to the measurement of transferrin, transthyretin, glycated hemoglobin, and homocysteine will be discussed, as will the limitations of the technology, and future directions for clinical protein measurement.