Tandem mass spectrometry (MS/MS or MS) is a widely used approach for structural annotation and identification of metabolites in complex biological samples. The importance of assessing the contribution of the precursor ion within an isolation window for MS experiments has been previously detailed in proteomics, where precursor ion purity influences the quality and accuracy of matching to mass spectral libraries, but to date, there has been little attention to this data-processing technique in metabolomics. Here, we present msPurity, a vendor-independent R package for liquid chromatography (LC) and direct infusion (DI) MS that calculates a simple metric to describe the contribution of the selected precursor. The precursor purity metric is calculated as "intensity of a selected precursor divided by the summed intensity of the isolation window". The metric is interpolated at the recorded point of MS acquisition using bordering full-scan spectra. Isotopic peaks of the selected precursor can be removed, and low abundance peaks that are believed to have limited contribution to the resulting MS spectra are removed. Additionally, the isolation efficiency of the mass spectrometer can be taken into account. The package was applied to Data Dependent Acquisition (DDA)-based MS metabolomics data sets derived from three metabolomics data repositories. For the 10 LC-MS DDA data sets with > ±1 Da isolation windows, the median precursor purity score ranged from 0.67 to 0.96 (scale = 0 to +1). The R package was also used to assess precursor purity of theoretical isolation windows from LC-MS data sets of differing sample types. The theoretical isolation windows being the same width used for an anticipated DDA experiment (±0.5 Da). The most complex sample had a median precursor purity score of 0.46 for the 64,498 XCMS determined features, in comparison to the less spectrally complex sample that had a purity score of 0.66 for 5071 XCMS features. It has been previously reported in proteomics that a purity score of <0.5 can produce unreliable spectra matching results. With this assumption, we show that for complex samples there will be a large number of metabolites where traditional DDA approaches will struggle to provide reliable annotations or accurate matches to mass spectral libraries.

In the fight against antibiotic resistance, drugs that target resistance mechanisms in bacteria can be used to restore the therapeutic effectiveness of antibiotics. The multidrug resistance efflux complex AcrAB-TolC is the most clinically relevant efflux pump in Enterobacterales and is a target for drug discovery. Inhibition of the pump protein AcrB allows the intracellular accumulation of a wide variety of antibiotics, effectively restoring their therapeutic potency. To facilitate the development of AcrB efflux inhibitors, it is desirable to discover the native substrates of the pump, as these could be chemically modified to become inhibitors. We analyzed the native substrate profile of AcrB in Escherichia coli MG1655 and Salmonella enterica serovar Typhimurium SL1344 using an untargeted metabolomics approach. We analyzed the endo- and exometabolome of the wild-type strain and their respective AcrB loss-of-function mutants (AcrB D408A) to determine the metabolites that are native substrates of AcrB. Although there is 95% homology between the AcrB proteins of S. Typhimurium and E. coli, we observed mostly different metabolic responses in the exometabolomes of the S. Typhimurium and E. coli AcrB D408A mutants relative to those in the wild type, potentially indicating a differential metabolic adaptation to the same mutation in these two species. Additionally, we uncovered metabolite classes that could be involved in virulence of S. Typhimurium and a potential natural substrate of AcrB common to both species. IMPORTANCE Multidrug-resistant Gram-negative bacteria pose a global threat to human health. The AcrB efflux pump confers inherent and evolved drug resistance to Enterobacterales, including Escherichia coli and Salmonella enterica serovar Typhimurium. We provide insights into the physiological role of AcrB: (i) we observe that loss of AcrB function in two highly related species, E. coli and S. Typhimurium, has different biological effects despite AcrB conferring drug resistance to the same groups of antibiotics in both species, and (ii) we identify potential natural substrates of AcrB, some of which are in metabolite classes implicated in the virulence of S. Typhimurium. Molecules that inhibit multidrug efflux potentiate the activity of old, licensed, and new antibiotics. The additional significance of our research is in providing data about the identity of potential natural substrates of AcrB in both species. Data on these will facilitate the discovery of, and/or could be chemically modified to become, new efflux inhibitors.

From the present study we conclude that a significant association between AA and AA metabolites in serum and PCa progression exists, although serum concentrations of HETEs exhibited low sensitivity toward the diagnosis of PCa.

Although many patients are cured from prostate cancer (PCa) by surgery only, there are still patients who will experience rising prostate-specific antigen (PSA) levels after surgery, a condition known as biochemical recurrence (BCR). Novel protein prognostic markers in PCa tissue might enable finding better treatment for those patients experiencing BCR with a high chance of metastasis. In this study, we aimed to identify altered proteins in prostate cancer tissue, and to evaluate their potential role as prognostic markers. We used two proteomics strategies to analyse 34 prostate tumours (PCa) and 33 normal adjacent prostate (NAP) tissues. An independent cohort of 481 samples was used to evaluate the expression of three proteins: AGR2, FASN and LOX5 as prognostic markers of the disease. Tissue microarray immunohistochemical staining indicated that a low percentage of positive tumour cells for AGR2 (HR (95% CI) = 0.61 (0.43-0.93)), and a low percentage of positive tumour cells for LOX5 expression (HR (95% CI) = 2.53 (1.23-5.22)) are predictors of BCR after RP. In contrast, FASN expression had no prognostic value for PCa.

Carboxylate‐substituted superhalogens of the type RCOOMX2− (M=Mg, Ca, Sr, Ba, Mn, Co, Ni, Zn; X=Cl, Br) are easily accessible in the gas phase by electrospray ionisation. Their collision‐induced dissociation (CID) characteristics have been probed by using ion‐trap and triple‐quadrupole mass analysers with particular emphasis on the behaviour of RCOOCaCl2−‐type ions. In the ion trap these appear to react readily with residual water to yield HOCaCl2− as the hydrolysis product. In the absence of water, a collision‐induced McLafferty‐type rearrangement takes over to produce HCaCl2− with the expulsion of an olefin and CO2. A brief computational analysis using the CBS‐QB3 model chemistry provides a satisfactory rationale for these observations. If complexed with MX2 (M=Mg, Ca, Sr, Ba), long‐chain unsaturated aliphatic carboxylate anions undergo various backbone cleavages upon collision. These lead to structure‐diagnostic olefin losses because the position of the double bonds remains intact. Such cleavages are absent in the bare ion RCOO−. The long‐chain ions RCOOMX2− also produce the intriguing species [CO2]MX2−.. These have been characterised by CID experiments, and theory indicates that they may be viewed as a CO2 molecule captured by the salt anion MX2−.. Finally, it is shown that the CID spectra of RCOOCaCl2− ions derived from all‐trans retinoic acid, a compound of current interest in biochemistry and medicine, show a unique structure‐diagnostic dissociation that may greatly aid its qualitative and quantitative analysis.