High-Throughput Metabolomics using 96-plex Isotope Tagging.

Liquid chromatography-mass spectrometry (LC-MS) based metabolomics suffers from extended duty cycles and matrix-dependent quantitation. Chemical tags with 96 unique masses are reported, which alleviate the metabolomic workflow bottleneck and allow for absolute quantitation. A metabolic screen for carboxylic acids was performed on mammalian cells deprived of various nutrients and showed 24% RSD and analysis of 288 samples in 2 h.

Discovery and Identification of Three Homocysteine Metabolites by Chemical Derivatization and Mass Spectrometry Fragmentation.

Discovery and identification of a new endogenous metabolite are typically hindered by requirements of large sample volumes and multistage purifications to guide synthesis of the standard. Presented here is a metabolomics platform that uses chemical tagging and tandem mass spectrometry to determine structure, direct synthesis, and confirm identity. Three new homocysteine metabolites are reported: N-succinyl homocysteine, 2-methyl-1,3-thiazinane-4-carboxylic acid (MTCA), and homolanthinone.

Double Cyclization Tandem Mass for Identification and Quantification of Phosphatidylcholines Using Isobaric Six-Plex Capillary nLC-MS/MS.

Multiplexing of phosphatidylcholine analysis is hindered by a lack of appropriate derivatization. Presented here is a tagging scheme that uses a quaternary amine tag and targets the hydroxy group of the phosphate, which switches the net charge from neutral to +2. Quantitative yields were achieved from >99% reaction completion derived by dimethoxymethyl morpholinium (DMTMM) activation.

Fluorescent human RPA to track assembly dynamics on DNA.

DNA metabolic processes including replication, repair, recombination, and telomere maintenance occur on single-stranded DNA (ssDNA). In each of these complex processes, dozens of proteins function together on the ssDNA template. However, when double-stranded DNA is unwound, the transiently open ssDNA is protected and coated by the high affinity heterotrimeric ssDNA binding Replication Protein A (RPA).

Fluorescent human RPA to track assembly dynamics on DNA.

DNA metabolic processes including replication, repair, recombination, and telomere maintenance occur on single-stranded DNA (ssDNA). In each of these complex processes, dozens of proteins function together on the ssDNA template. However, when double-stranded DNA is unwound, the transiently open ssDNA is protected and coated by the high affinity heterotrimeric ssDNA binding Replication Protein A (RPA).

Influence of circadian phase and extended wakefulness on glucose levels during forced desynchrony.

Objectives: Circadian misalignment and sleep deprivation often occur in tandem, and both negatively impact glucose homeostasis and metabolic health. The present study employed a forced desynchrony protocol to examine the influence of extended wakefulness and circadian misalignment on hourly glucose levels.

Methods: Nine healthy adults (4F/5M; 26 ± 4years) completed a 31-day in-laboratory protocol.

Dual Fragmentation Isobaric Tags for Metabolomics.

Isobaric tags typically leverage an a1 type fragmentation to produce constant mass reporter ions. While this motif allows for efficient reporter formation, isobaric tags lack structural diversity, which limits the number and type of isotopes that are synthetically available. Presented here are two examples of dual fragmentation isobaric tagging.

Isobaric 6-plex and tosyl dual tagging for the determination of positional isomers and quantitation of monounsaturated fatty acids using rapid UHPLC-MS/MS.

Isobaric labelling of fatty acids is complicated by chromatographic co-elution of double bond isomers. This produces contaminated spectra which can mask important biological changes. Here two derivatization strategies are combined to improve throughput and produce MS reporters which change mass depending on double bond position.

Isobaric 4-Plex Tagging for Absolute Quantitation of Biological Acids in Diabetic Urine Using Capillary LC-MS/MS.

Isobaric labeling in mass spectrometry enables multiplexed absolute quantitation and high throughput, while minimizing full scan spectral complexity. Here, we use 4-plex isobaric labeling with a fixed positive charge tag to improve quantitation and throughput for polar carboxylic acid metabolites. The isobaric tag uses an isotope-encoded neutral loss to create mass-dependent reporters spaced 2 Da apart and was validated for both single- and double-tagged analytes.

Neutron encoded derivatization of endothelial cell lysates for quantitation of aldehyde metabolites using nESI-LC-HRMS.

Biological aldehydes are difficult to analyze by electrospray ionization mass spectrometry due to their poor proton affinity and low biological concentrations. Chemical derivatization with stable isotope tags is used here for sample multiplexing, increased throughput, improved signal intensity, and quantitation. Nine quaternary amine tags with mass differences as low as 0.

Effects of photodeoxygenation on cell biology using dibenzothiophene S-oxide derivatives as O(P)-precursors.

Photodeoxygenation of dibenzothiophene S-oxide and its derivatives have been used to generate atomic oxygen [O(P)] to examine its effect on proteins, nucleic acids, and lipids. The unique reactivity and selectivity of O(P) have shown distinct oxidation products and outcomes in biomolecules and cell-based studies. To understand the scope of its global impact on the cell, we treated MDA-MB-231 cells with 2,8-diacetoxymethyldibenzothiophene S-oxide and UV-A light to produce O(P) without targeting a specific cell organelle.

Selective estrogen receptor modulator, tamoxifen, inhibits Zika virus infection.

Zika virus (ZIKV) is an arbovirus belonging to the flaviviridae family with a risk assessment that has been increasing in recent years and was labeled a global health emergency by the World Health Organization in 2016. There are currently no Food and Drug Administration-approved treatment options available for ZIKV, so expeditious development of treatment options is urgent. To expedite this process, an on-market drug, tamoxifen (TAM), was selected as a promising candidate for repurposing due to its wide range of biological activities and because it has already been shown to possess activity against hepatitis C virus, a flavivirus in a separate genus.

Synthesis of triphenylphosphonium dibenzothiophene S-oxide derivatives and their effect on cell cycle as photodeoxygenation-based cytotoxic agents.

Photodeoxygenation of Dibenzothiophene-S-oxide (DBTO) in UV-A light produces atomic oxygen [O(P)] and the corresponding sulfide, dibenzothiophene (DBT). Recently, DBTO has been derivatized to study the effect of UV-A light-driven photodeoxygenation in lipids, proteins, and nucleic acids. In this study, two DBTO derivatives with triphenylphosphonium groups were synthesized to promote mitochondrial accumulation.

Dibenzothiophene Sulfone Derivatives as Plasma Membrane Dyes.

Dibenzothiophene 5,5-dioxide (DBTOO) derivatives have recently been shown to processes utility as fluorescent cell dyes. In an effort to extend the functionality of DBTOO-based dyes to include the visualization of cellular membranes, two lipophilic DBTOO were synthesized and their ability to incorporate into the plasma membrane of HeLa cells was examined by fluorescent microscopy. The photophysical properties of the two new DBTOO derivatives were determined and both have good fluorescent quantum yields and a visible blue emission.

Effect of modafinil on impairments in neurobehavioral performance and learning associated with extended wakefulness and circadian misalignment.

Although worldwide millions of people work prolonged hours, at adverse circadian phases, evidence suggests that cognitive function is impaired under these conditions with important societal consequences. In a double-blind placebo-controlled laboratory-based study, we investigated the effect of the wakefulness-promoting drug modafinil as a countermeasure against such neurobehavioral impairments induced by both prolonged wakefulness and circadian misalignment. Neurobehavioral performance, alertness, and sleep were studied in young healthy participants (N=18) who underwent a 25-day forced desynchrony protocol in which the period of the sleep-wakefulness cycle was scheduled to be 42.

Diurnal variation in CSF orexin-A in healthy male subjects.

Study Objective: Orexin-A is hypothesized to promote wakefulness, and we examined whether cerebrospinal fluid (CSF) orexin-A levels are higher during the waking period in man.

Design: Within-subjects, repeated-measures design with balanced ordering of sampling at approximately 5 AM and 5 PM.

Participants: Eight healthy young males.

Partial rescue of the Hyp phenotype by osteoblast-targeted PHEX (phosphate-regulating gene with homologies to endopeptidases on the X chromosome) expression.

Inactivating mutations and/or deletions of PHEX/Phex (phosphate-regulating gene with homologies to endopeptidases on the X chromosome) are responsible for X-linked hypophosphatemic rickets in humans and in the murine homolog Hyp. The predominant osteoblastic expression of Phex has implicated a primary metabolic osteoblast defect in the pathophysiology of this disorder. By targeting PHEX expression to osteoblasts in the Hyp genetic background, we aimed to correct the corresponding biochemical and morphological abnormalities and obtain information on their pathogenetic mechanism.