Untargeted urine metabolite profiling by mass spectrometry aided by multivariate statistical analysis to predict prostate cancer treatment outcome.

Deciphering metabolomic networks has been demonstrated to provide valuable information for diagnosing and monitoring diseases. Herein, we report a technique to monitor untargeted urine metabolites to evaluate prostate cancer aggressiveness and treatment outcome. Direct chemical profiling of urine was achieved by a combined procedure of hyphenating laser diode thermal desorption with atmospheric pressure chemical ionization mass spectrometry (LDTD-APCI-MS). We describe a conceptually new approach to monitoring preoperative urinary metabolic alterations associated with prostate cancer recurrence. By evaluating mass/charge (m/z) ratios and peak intensities of ions detected by mass spectroscopy of urine samples, we revealed that intensities at m/z 313.2740 (±0.0003) and 341.3054 (±0.0006) attributable to monoacylglycerol backbone fragments from glycerides can be statistically correlated to disease progression.

Primary Metabolism co-Opted for Defensive Chemical Production in the Carabid Beetle, Harpalus pensylvanicus.

Of the approximately one million described insect species, ground beetles (Coleoptera: Carabidae) have long captivated the attention of evolutionary biologists due to the diversity of defensive compounds they synthesize. Produced using defensive glands in the abdomen, ground beetle chemicals represent over 250 compounds including predator-deterring formic acid, which has evolved as a defensive strategy at least three times across Insecta. Despite being a widespread method of defense, formic acid biosynthesis is poorly understood in insects. Previous studies have suggested that the folate cycle of one-carbon (C1) metabolism, a pathway involved in nucleotide biosynthesis, may play a key role in defensive-grade formic acid production in ants. Here, we report on the defensive gland transcriptome of the formic acid-producing ground beetle Harpalus pensylvanicus. The full suite of genes involved in the folate cycle of C1 metabolism are significantly differentially expressed in the defensive glands of H. pensylvanicus when compared to gene expression profiles in the rest of the body. We also find support for two additional pathways potentially involved in the biosynthesis of defensive-grade formic acid, the kynurenine pathway and the methionine salvage cycle. Additionally, we have found an array of differentially expressed genes in the secretory lobes involved in the biosynthesis and transport of cofactors necessary for formic acid biosynthesis, as well as genes presumably involved in the detoxification of secondary metabolites including formic acid. We also provide insight into the evolution of the predominant gene family involved in the folate cycle (MTHFD) and suggest that high expression of folate cycle genes rather than gene duplication and/or neofunctionalization may be more important for defensive-grade formic acid biosynthesis in H. pensylvanicus. This provides the first evidence in Coleoptera and one of a few examples in Insecta of a primary metabolic process being co-opted for defensive chemical biosynthesis. Our results shed light on potential mechanisms of formic acid biosynthesis in the defensive glands of a ground beetle and provide a foundation for further studies into the evolution of formic acid-based chemical defense strategies in insects.

Biosynthetic origin of benzoquinones in the explosive discharge of the bombardier beetle Brachinus elongatulus.

Bombardier beetles are well-known for their remarkable defensive mechanism. Their defensive apparatus consists of two compartments known as the reservoir and the reaction chamber. When challenged, muscles surrounding the reservoir contract sending chemical precursors into the reaction chamber where they mix with enzymes resulting in an explosive discharge of a hot noxious chemical spray containing two major quinones: 1,4-benzoquinone and 2-methyl-1,4-benzoquinone (toluquinone). Previously, it has been speculated that the biosynthesis of all benzoquinones originates from one core precursor, 1,4-hydroquinone. Careful ligation of the base of the reservoir chamber enabled us to prevent the explosive reaction and sample untransformed reservoir fluid, which showed that it accumulates significant quantities of 1,4-hydroquinone and 2-methyl-1,4-hydroquinone. We investigated the biosynthetic mechanisms leading to quinone formation by injecting or feeding Brachinus elongatulus beetles with stable-isotope-labeled precursors. Chemical analysis of defensive secretion samples obtained from 1,4-hydroquinone-d6-administered beetles demonstrated that it underwent conversion specifically to 1,4-benzoquinone. Analogously, results from m-cresol-d8 injected or fed beetles confirmed that m-cresol is metabolized to 2-methyl-1,4-hydroquinone, which is then oxidized to 2-methyl-1,4-benzoquinone in the hot spray. Our results refute the previous claim that 1,4-hydroquinone is the precursor of all substituted benzoquinones in bombardier beetles and reveal that they are biosynthetic products of two independent pathways. Most likely, the aforementioned biosynthetic channel of hydroxylation of appropriate phenolic precursors and subsequent oxidation is not restricted to bombardier beetles; it could well be a general pathway that leads to the formation of all congeners of benzoquinones, one of the most widely distributed groups of defensive compounds in arthropods. Graphical abstract.

HCN emission by a Polydesmid Millipede Detected Remotely by Reactive Adsorption on Gold Nanoparticles Followed by Laser Desorption/Ionization Mass Spectrometry (LDI-MS).

Hydrocyanic acid (HCN) is a well-known defensive allomone in the chemical arsenal of millipedes in the order Polydesmida. The presence of HCN in the headspace vapor of adult Xystocheir dissecta (Wood, 1867), a common millipede from the San Francisco Bay Area, was traced by laser desorption/ionization-mass spectrometry (LDI-MS). To accomplish this, the headspace vapor surrounding caged, live millipedes was allowed to diffuse passively over gold-nanoparticle (AuNP) deposits placed at various distances from the emitting source. The stainless steel plates with AuNP deposits were removed and irradiated by a 355-nm laser. The gaseous ions generated in this way were detected by time-of-flight mass spectrometry. The intensity of the mass spectrometric peak detected at m/z 249 for the Au(CN)2- complex anion was compared to that of the residual Au- signal (m/z 197). Using this procedure, HCN vapors produced by the live millipedes could be detected up to 50 cm away from the source. Furthermore, the addition of H2O2, as an internal oxygen source for the gold cyanidation reaction that takes place in the AuNP deposits, significantly increased the detection sensitivity. Using the modified H2O2 addition procedure, HCN could now be detected at 80 cm from the source. Moreover, we found a decreasing intensity ratio of the Au(CN)2-/Au- signals as the distance from the emitting source increased, following an exponential-decay distribution as predicted by Fick's law of diffusion. Graphical abstract.

Identification of alkylpyrazines by gas chromatography mass spectrometry (GC-MS).

Pyrazines are an important group of natural products widely used as food additives and fragrants. Gas chromatography-mass spectrometry (GCMS) is the most widely applied analytical technique for characterization of alkylpyrazines. However, mass spectra of many positional isomers of alkylpyrazines are very similar. Consequently, an unambiguous identification of each positional isomer by spectral interpretation or database search protocols is practically unfeasible. In fact, there are many misidentifications in literature. To identify alkylpyrazines, chemists often resort to gas chromatographic retention indices (RIs). Although there are many compilations of retention indices of alkylpyrazines, these databases are often incomplete and the values reported are sometimes inconsistent. Herein, we present retention indices of fifty-six alkylpyrazines recorded on DB-1, ZB-5MS, DB-624, and ZB-WAXplus stationary phases, and compare them with those available in the NIST-2017 MS-RI database. Furthermore, we demonstrate how RI values can be used, together with mass spectral interpretations, to identify certain alkylpyrazines unambiguously.

Gas-phase protomers of p-(dimethylamino)chalcone investigated by travelling-wave ion mobility mass spectrometry (TWIMS).

Results from ion-mobility (IM) separation experiments demonstrate that O- and N-protomers of p-(dimethylamino)chalcone (p-DMAC) can coexist in the gas phase. The relative populations of the two protomers strongly depend on the ion-generating settings and the conditions the precursor ions experience from the point of their gas-phase inception to the time of their detection. Under relatively dry source conditions, the ratio of the gas-phase protomers generated under helium-plasma ionization (HePI) conditions is biased towards the thermodynamically favored O-protomer. However, when the humidity of the enclosed ion source was increased, the IM arrival-time distribution profile of the mass-selected protonated precursor of p-DMAC changed rapidly to one dominated by the N-protomer. Under spray-ionization conditions, the formation of the thermodynamically less favored protomer has been generally attributed to a phenomenon called kinetic trapping. Herein, we demonstrate that the population of thermodynamically less favored N-protomer can be dramatically increased simply by introducing water vapor to the HePI ion source.

Alkyl-Dimethylpyrazines in Mandibular Gland Secretions of Four Odontomachus Ant Species (Formicidae: Ponerinae).

Ponerine ants are known to contain mixtures of pyrazines in their mandibular glands. We analyzed the mandibular gland contents of four ponerine species (Odontomachus chelifer, O. erythrocephalus, O. ruginodis, and O. bauri) by gas chromatography coupled with mass spectrometry, and found that each species contains specific mixtures of trisubstituted alkylpyrazines among other volatiles. Attempts to identify alkylpyrazines solely by mass spectral interpretation is unrealistic because spectra of positional isomers are indistinguishable. To avoid misidentifications, we synthesized a large number of reference compounds and compared their mass spectral and gas chromatographic properties with those present in the Odontomachus species under investigation. Most of the compounds identified were 2-alkyl-3,5-dimethylpyrazines. Interestingly, when the third substituent was an isopentyl group, the two methyl groups were found to be located at the 2 and 5 ring positions. Using our data, we recognized several misidentifications in previous publications.

Collision-induced dissociation processes of protonated benzoic acid and related compounds: competitive generation of protonated carbon dioxide or protonated benzene.

Upon activation in the gas phase, protonated benzoic acid (m/z 123) undergoes fragmentation by several mechanisms. In addition to the predictable water loss followed by a CO loss, the m/z 123 ion more intriguingly eliminates a molecule of benzene to generate protonated carbon dioxide (H - O+ ═ C ≡ O, m/z 45), or a molecule of carbon dioxide to yield protonated benzene (m/z 79). Experimental evidence shows that the incipient proton ambulates during the fragmentation processes. For the CO2 or benzene loss, protonated benzoic acid transfers the charge-imparting proton initially to the ortho position and then to the ipso position to generate a transient species which dissociates to form an ion-neutral complex between benzene and protonated CO2 . The formation of the m/z 45 ion is not a phenomenon unique to benzoic acid: spectra from protonated isophthalic acid, terephthalic acid, trans-cinnamic acid and some aliphatic acids also displayed a peak for m/z 45. However, the m/z 45 peak is structurally diagnostic only for certain benzene polycarboxylic acids because the spectra of compounds with two carboxyl groups on adjacent ring carbons do not produce a peak at m/z 45. For the m/z 79 ion to be formed, an intramolecular reaction should take place in which protonated CO2 within the ion-neutral complex acts as the attacking electrophile to transfer a proton to benzene. Copyright © 2017 John Wiley & Sons, Ltd.

Ambulation of Incipient Proton during Gas-Phase Dissociation of Protonated Alkyl Dihydrocinnamates.

Upon activation in the gas phase, protonated alkyl dihydrocinnamates undergo an alcohol loss. However, the mechanism followed is not a simple removal of an alkanol molecule after a protonation on the alkoxy group. The mass spectrum of the m/z 166 ion for deuteron-charged methyl dihydrocinnamate showed two peaks of 1:5 intensity ratio at m/z 133 and 134 to confirm that the incipient proton is mobile. The proton initially attached to the carbonyl group migrates to the ring and randomizes before a subsequent transfer of one of the ring protons to the alkoxy group for the concomitant alcohol elimination. Moreover, protonated methyl dihydrocinnamate undergoes more than one H/D exchange. The spectra recorded from m/z 167 and 168 ions obtained for di- and tri-deuterio isotopologues showed peak pairs at m/z 134, 135 and 135, 136, at 1:2 and 1:1 intensity ratios, respectively, confirming the benzenium ion intermediate achieves complete randomization before the proton transfer. Additionally, protonated higher esters of alkyl dihydrocinnamates undergo a cleavage of the O-CH2 bond to form an ion/neutral complex, which, upon activation, dissociates generating a carbenium ion and dihydrocinnamic acid, or rearranges to generate protonated dihydrocinnamic acid and an alkene by a nonspecific proton transfer.