Untargeted Method Research Articles

An extendable all-in-one injection twin derivatization LC-MS/MS strategy for the absolute quantification of multiple chemical-group-based submetabolomes

The ability of LC-MS/MS for high coverage metabolite analysis lags behind the requirements of global metabolomics. The introduction of chemical derivatizations could significantly extend the ability of LC-MS/MS with enhanced MS response and improved LC separation, which has been serving as a promising quantitative tool for metabolomic analysis. However, as one specific derivatization reagent usually targets to a certain moiety, only a single chemical-group-based submetabolome could be analyzed in one injection. Therefore, the coverage of detected metabolites by derivatization-based LC-MS/MS is largely limited. To overcome this technical obstacle of derivatization-based LC-MS and increase submetabolome coverage, we proposed an extendable all-in-one injection LC-MS/MS strategy. 5-dimethylamino-naphthalene-1-sulfonyl chloride (Dns-Cl)/5-diethylamino-naphthalene-1-sulfonyl chloride (Dens-Cl) and 5-dimethylamino-naphthalene-1-sulfonyl piperazine (Dns-PP)/5-diethylamino-naphthalene-1-sulfonyl piperazine (Dens-PP) were used as twins labeling reagents for amino/phenol and carboxyl submetabolomes, respectively. “Series Mode” and “Parallel Mode” were proposed and investigated using eight representative standards with the consideration of interaction between different derivatization systems, time-consumption, and extendability. As a result, we found that “Series Mode” led to yield reduction, while “Parallel Mode” gave identical results with those of individual derivatization. Finally, a “Parallel Mode” was chosen to develop an extendable all-in-one injection twin derivatization LC-MS/MS strategy to quantify eighty metabolites assigned to five classes of microbial metabolites, including polyamines, amino acids, indole derivatives, bile acids, and free fatty acids. This well-validated method quantified 67 metabolites absolutely and discovered additional 40 differential metabolites compared with the untargeted method in rat serum from irinotecan (CPT-11)-induced gastrointestinal toxicity model.

Rapid untargeted screening for drug residues in animal tissues with liquid microjunction surface sampling probe mass spectrometry

An untargeted screening method for the rapid identification of veterinary drug residues in incurred animal tissues using liquid microjunction surface sampling probe mass spectrometry (LMJSSP-MS) was developed. Current analytical methods for veterinary drug residue screening involve lengthy sample preparation, extraction, and instrumental analysis steps. This method identifies veterinary drug residues in several different incurred animal tissues more quickly than conventional analytical methods. This LMJSSP-MS method uses an ambient ionization technology called liquid microjunction surface sampling probe along with a data dependent scan function of a quadrupole orbitrap mass spectrometer. Collected product ion spectra are searched against the mzCloud™ online mass spectral database to identify veterinary drug residues found in incurred animal tissue samples. Examples of veterinary drugs identified with this method include flunixin, tilmicosin, pentobarbital, xylazine, and ketamine. Optimization of method parameters is described and discussed. The limit of identification (LOI) of this method is estimated to be approximately 1 μg g−1 for xylazine and ketamine.

Bretschneider solution-induced alterations in the urine metabolome in cardiac surgery patients

The development of Bretschneider’s histidine-tryptophan-ketoglutarate (HTK) cardioplegia solution represented a major advancement in cardiac surgery, offering significant myocardial protection. However, metabolic changes induced by this additive in the whole body have not been systematically investigated. Using an untargeted mass spectrometry-based method to deeply explore the urine metabolome, we sought to provide a holistic and systematic view of metabolic perturbations occurred in patients receiving HTK. Prospective urine samples were collected from 100 patients who had undergone cardiac surgery, and metabolomic changes were profiled using a high-performance chemical isotope labeling liquid chromatography-mass spectrometry (LC-MS) method. A total of 14,642 peak pairs or metabolites were quantified using differential 13C-/12C-dansyl labeling LC-MS, which targets the amine/phenol submetabolome from urine specimens. We identified 223 metabolites that showed significant concentration change (fold change > 5) and assembled several potential metabolic pathway maps derived from these dysregulated metabolites. Our data indicated upregulated histidine metabolism with subsequently increased glutamine/glutamate metabolism, altered purine and pyrimidine metabolism, and enhanced vitamin B6 metabolism in patients receiving HTK. Our findings provide solid evidence that HTK solution causes significant perturbations in several metabolic pathways and establish a basis for further study of key mechanisms underlying its organ-protective or potential harmful effects.

Development of a sensitive untargeted liquid chromatography-high resolution mass spectrometry screening devoted to hair analysis through a shared MS2 spectra database: A step toward early detection of new psychoactive substances.

Untargeted liquid chromatography-high resolution mass spectrometry (LC-HRMS) techniques have become indispensable tools for systematic toxicological analysis. They allow the research of an almost unlimited number of drugs within a single analytical cycle, but shared mass spectra libraries are still missing to identify newly marketed compounds, along with defined analytical procedures. This article describes the optimization, validation, and application of an untargeted screening method devoted to hair analysis using data-dependent analysis (DDA) and a shared HRMS database. This method used an ultra-high performance liquid chromatography coupled to a benchtop Orbitrap. Raw MS data were processed with Compound Discoverer software coupled to the mzCloud™ library. Optimizations were performed on blank hair spiked with 19 analytes having different physical and chemical properties. To validate the effectiveness of a shared spectra database, 20 compounds spectra were added and then retrospectively screened. Sensitivity and reliability were evaluated on 317 compounds of interest in toxicology. The method was then applied to 11 hair samples. The matrix effect range by ion suppression/enhancement was 40%-110%. The method allows the detection of 284 among the 317 screened compounds, including 72 new psychoactive substances (NPS). Lower limit of identification (LLOI) and lower limit of detection (LLOD) were 1 to 1000pg/mg and 1 to 500pg/mg, respectively. The method was successfully applied to 11 clinical cases and 144 compounds were identified including 24 NPS including AKB48-5F for the first time in hair. We developed and validated an LC-HRMS untargeted screening of 284 compounds and successfully applied it to 11 real hair samples.

Targeted and Untargeted Detection of DNA Adducts of Aromatic Amine Carcinogens in Human Bladder by Ultra-Performance Liquid Chromatography-High-Resolution Mass Spectrometry.

Epidemiological studies have linked aromatic amines (AAs) from tobacco smoke and some occupational exposures with bladder cancer risk. Several epidemiological studies have also reported a plausible role for structurally related heterocyclic aromatic amines present in tobacco smoke or formed in cooked meats with bladder cancer risk. DNA adduct formation is an initial biochemical event in bladder carcinogenesis. We examined paired fresh-frozen (FR) and formalin-fixed paraffin-embedded (FFPE) nontumor bladder tissues from 41 bladder cancer patients for DNA adducts of 4-aminobiphenyl (4-ABP), a bladder carcinogen present in tobacco smoke, and 2-amino-9 H-pyrido[2,3- b]indole, 2-amino-1-methyl-6-phenylimidazo[4,5- b]pyridine and 2-amino-3,8-dimethylimidazo[4,5- f]quinoxaline, possible human carcinogens, which occur in tobacco smoke and cooked meats. These chemicals are present in urine of tobacco smokers or omnivores. Targeted DNA adduct measurements were done by ultra-performance liquid chromatography-electrospray ionization multistage hybrid Orbitrap MS. N-(2'-Deoxyguanosin-8-yl)-4-ABP ( N-(dG-C8)-4-ABP) was the sole adduct detected in FR and FFPE bladder tissues. Twelve subjects (29%) had N-(dG-C8)-4-ABP levels above the limit of quantification, ranging from 1.4 to 33.8 adducts per 109 nucleotides (nt). DNA adducts of other human AA bladder carcinogens, including 2-naphthylamine (2-NA), 2-methylaniline (2-MA), 2,6-dimethylaniline (2,6-DMA), and lipid peroxidation (LPO) adducts, were screened for in bladder tissue, by our untargeted data-independent adductomics method, termed wide-selected ion monitoring (wide-SIM)/MS2. Wide-SIM/MS2 successfully detected N-(dG-C8)-4-ABP, N-(2'-deoxyadenosin-8-yl)-4-ABP and the presumed hydrazo linked adduct, N-(2'-deoxyguanosin- N2-yl)-4-ABP, and several LPO adducts in bladder DNA. Wide-SIM/MS2 detected multiple DNA adducts of 2-NA, 2-MA, and, 2,6-DMA, when calf thymus DNA was modified with reactive intermediates of these carcinogens. However, these AA-adducts were below the limit of detection in unspiked human bladder DNA (<1 adduct per 108 nt). Wide-SIM/MS2 can screen for many types of DNA adducts formed with exogenous and endogenous electrophiles and will be employed to identify DNA adducts of other chemicals that may contribute to the etiology of bladder cancer.

An integrative UHPLC-MS/MS untargeted metabonomics combined with quantitative analysis of the therapeutic mechanism of Si-Ni-San

A UHPLC-MS/MS untargeted serum metabonomic method combined with quantitative analysis of five potential biomarkers in rat serum was developed and validated, to further understand the anti-liver injury effect of Si-Ni-San and its mechanism on liver injury rats in this study. The metabolites were separated and identified on BEH C18 column (100 mm × 2.1 mm, 1.7 μm) using the ACQUITY UHPLC-MS system (Waters Corp., Milford, MA, USA). Principal component analysis (PCA) was used to identify potential biomarkers. Primary potential biomarkers including phenylalanine, tryptophan, Glycochenodeoxycholic acid (GCDCA) and hysophosphatidylcholine (LPC), which were related to amino acid metabolism, lipid metabolism, bile acid biosynthesis and oxidation-antioxidation balance, were found in the untargeted metabonomic research. Moreover, these targeted biomarkers were further separated and quantified in multiple-reaction monitoring (MRM) with positive ionization mode. The proposed method was linear for each analyte with correlation coefficients over 0.99. The intra- and inter-day precision values (relative standard deviation, RSD) were less than 13.1% and accuracy (relative error, RE) was from −9.5% to 10.3% at all quality control (QC) levels. The validated method was successfully applied to study the serum samples of control group, model group, positive control group (silymarin group) and Si-Ni-San group in rats.

Discrimination of geographical origin of oranges (Citrus sinensis L. Osbeck) by mass spectrometry-based electronic nose and characterization of volatile compounds

An untargeted method using headspace solid-phase microextraction coupled to electronic nose based on mass spectrometry (HS-SPME/MS-eNose) in combination with chemometrics was developed for the discrimination of oranges of three geographical origins (Italy, South Africa and Spain). Three multivariate statistical models, i.e. PCA/LDA, SELECT/LDA and PLS-DA, were built and relevant performances were compared. Among the tested models, SELECT/LDA provided the highest prediction abilities in cross-validation and external validation with mean values of 97.8% and 95.7%, respectively. Moreover, HS-SPME/GC-MS analysis was used to identify potential markers to distinguish the geographical origin of oranges. Although 28 out of 65 identified VOCs showed a different content in samples belonging to different classes, a pattern of analytes able to discriminate simultaneously samples of three origins was not found. These results indicate that the proposed MS-eNose method in combination with multivariate statistical analysis provided an effective and rapid tool for authentication of the orange's geographical origin.

Interlaboratory Coverage Test on Plant Food Bioactive Compounds and their Metabolites by Mass Spectrometry-Based Untargeted Metabolomics.

Bioactive compounds present in plant-based foods, and their metabolites derived from gut microbiota and endogenous metabolism, represent thousands of chemical structures of potential interest for human nutrition and health. State-of-the-art analytical methodologies, including untargeted metabolomics based on high-resolution mass spectrometry, are required for the profiling of these compounds in complex matrices, including plant food materials and biofluids. The aim of this project was to compare the analytical coverage of untargeted metabolomics methods independently developed and employed in various European platforms. In total, 56 chemical standards representing the most common classes of bioactive compounds spread over a wide chemical space were selected and analyzed by the participating platforms (n = 13) using their preferred untargeted method. The results were used to define analytical criteria for a successful analysis of plant food bioactives. Furthermore, they will serve as a basis for an optimized consensus method.

Peptide biomarkers for identifying the species origin of gelatin using coupled UPLC-MS/MS

Liquid chromatography linked with mass spectrometry (LC–MS/MS) was used to analyse gelatin from four different species after a trypsin digest. Using chemometric software to analyse the data it was possible to find peptide fragments that were specific to each species of gelatin: porcine, bovine, chicken or fish. Identification of these peptides was challenging due to the destructive nature of gelatin manufacture. The untargeted workflow method developed allowed identification of 21 unknown gelatin samples with 100% accuracy. Fish gelatin is made from a large range of different species that do not share a common differentiating protein but it was shown that the protein from a parasitic bacteria could be used to identify fish gelatin.

Multiclass screening in urine by comprehensive two-dimensional liquid chromatography time of flight mass spectrometry for residues of sulphonamides, beta-agonists and steroids

ABSTRACTNowadays routine residue monitoring involves the analysis of many compounds from different classes, mainly in urine. In the past two decades, developments heavily focused on the use of mass spectrometers (MS) and faster and more sensitive MS detectors have reached the market. However, chromatographic separation (CS) was rather ignored and the cognate developments in CS were not in line. As a result, residue analysis did not improve to the extent anticipated. CS by LC x LC is a promising technique and will enable a further increase in the range of compounds and compound classes that can be detected in a single run. In the present study, a self-built LC x LC system, using a 10 port valve, was connected to a single quadrupole MS with electrospray interface. Standards containing a mixture of sulphonamides, β-agonists and (steroid) hormones, 53 compounds, in total, were analysed. Results demonstrated that these compounds were well separated and could be detected at low levels in urine, i.e. limit of detection (LOD) from 1 µg L−1 for most β-agonists to 10 µg L−1 for some sulphonamides and most hormones. To enhance the sensitivity, optimisation was performed on an advanced commercial LC x LC system connected to a full scan accurate MS. This ultimately resulted in a fast high throughput untargeted method, including a simple sample clean-up in a 96-well format, for the analysis of urine samples.

New application of direct analysis in real time high-resolution mass spectrometry for the untargeted analysis of fresh and aged secondary organic aerosols generated from monoterpenes.

Secondary organic aerosols (SOAs) represent a significant portion of total atmospheric aerosols. They are generated by the oxidation of volatile organic compounds (VOCs), and particularly biogenic VOCs (BVOCs). The analysis of such samples is usually performed by targeted methods that often require time-consuming preparation steps that can induce loss of compounds and/or sample contaminations. Recently, untargeted methods using high-resolution mass spectrometry (HRMS) have been successfully employed for a broad characterization of chemicals in SOAs. Herein we propose a new application of the direct analysis in real time (DART) ionization method combined with HRMS to quickly detect several hundred chemicals in SOAs collected on a quartz filter without sample preparation or separation techniques. The reproducibility of measurements was good, with several hundred elemental compositions common to three different replicates. The relative standard deviations of the intensities of the chemical families ranged from 6% to 35%, with sufficient sensitivity to allow the unambiguous detection of 4 ng/mm2 of pinic acid. The presence of oligomers and specific tracers was highlighted by MSn (n ≤ 4) experiments, an achievement that is difficult to attain with other ultrahigh-resolution mass spectrometers. Contributions of this untargeted DART-HRMS method were illustrated by the analysis of fresh and aged SOAs from different gaseous precursors such as limonene, a β-pinene/limonene mixture or scots pines emissions. The results show that it is possible to use DART-HRMS for the identification of tracers of specific aging reactions, or for the identification of aerosols from specific biogenic precursors.

Diagnostic value of trans rectal ultra-sonography in comparison with MR imaging in detection and characterization of prostatic lesions

Aim:To evaluate the diagnostic accuracy of MRI techniques in detection and characterization of different prostatic lesions in comparison with TRUS. Patient and Methods:This prospective study include TRUS and MRI prostate of 30 adult male patients presented by different prostatic lesions obtained by using 1.5Tmachine, using pelvic phased array coil and/or endorectal coil. Pulse sequences include conventional (T1WT serum prostate-specific antigen (PSA), a nonspecific blood test; and Trans rectal ultrasound (TRUS)–guided biopsy, a standardized but untargeted method. Because of the limitations of these available diagnostic tools, advances in MRI techniques show potential for improving the diagnostic accuracy of MRI for prostatic lesions detection. A recently developed Multiparametric MRI approach that combines anatomic T2-weighted imaging with functional data appears to be one of the most promising techniques for prostatic lesions detection

Exploring lipid markers of the quality of coix seeds with different geographical origins using supercritical fluid chromatography mass spectrometry and chemometrics

Background: Lipids, a group of primary metabolites, could be used as quality markers of Traditional Chinese medicine.Purpose: The present study was designed to develop a research method to explore lipid markers of the quality of coix seeds with different geographical origins.Study Design: The geographical origins of coix seeds were divided into three regions based on the latitude. A central composite design (CCD test) was used to optimize the chromatographic parameters of supercritical fluid chromatography to obtain optimal lipid profile of coix seed.Methods: An untargeted method based on ultra-performance convergence chromatography - quadrupole/time-of-flight hybrid mass spectrometry (UPC2-QTOF) was developed. Four chromatographic parameters were optimized using CCD test, and a fusion index established by Derringer function was used to evaluate. The lipid profile of 27 batches of coix seeds were acquired and processed by Progenesis QI software, and the MS/MS spectrums were obtained to identify, simultaneously. The difference lipids were explored by orthogonal partial least squares discriminant analysis (OPLS-DA). The lipids that showed differences depending on their seeds’ geographical origin were selected as markers of the quality of coix seeds from the three regions.Results: A Torus 2-PIC (1.7 µm, 100 mm × 3.0 mm) was selected as the optimal column of the untargeted method which the run time was only 8 minutes. From the CCD test, the interaction of chromatographic parameters between column temperature and backpressure was founded which the optimal parameters were 55 °C and 2600 psi, respectively. Thirty-two peaks in the lipid profile of coix seed were tentatively identified, of which 20 were triglyceride, and 12 were diglyceride. Nine features that could potentially be used to distinguish the coix seeds by their geographical origin were identified, most of which were diglycerides, such as OP.Conclusions: Our findings confirm that UPC2-QTOF combined with chemometrics could be used as an efficient method for exploring potential lipid markers of the quality of herbal medicine.

Metabonomics Approach To Comparing the Antistress Effects of Four Panax ginseng Components in Rats.

Different components of Panax ginseng have different properties and medicinal effects. Metabonomics was a prospective approach to analyze the global response of endogenous metabolites to physiological and pathological processes. In this study, an untargeted metabonomics method using GC/TOFMS combined with multivariate statistical techniques was applied to compare entire metabolite differences and the antistress variations among four components of P. ginseng, namely, total ginsenosides (TG), panaxadiol (PD), panaxatriol (PT), and ginseng polysaccharide (PS), in Wistar rats. The results of metabolite analysis showed that numerous urine metabolites involving neurotransmitters, amino acids, organic acids, and gut microbiota metabolites were changed after administration of the four components of P. ginseng, with TG having the least impact on urinary metabolites. The urinary metabolite profiling of these rats exposed to acute combined stress (forced swimming and behavior restriction) demonstrated that the four ginseng components attenuated urine metabolite changes involving gut microbiota metabolites, tricarboxylic acid (TCA) cycle and energy metabolites, and organic acids to different degrees, with TG improving most of the metabolites altered by stress.

Dissociation mechanisms-based UHPLC Q-Orbitrap strategy for screening of cephalosporins and metabolites in shrimp

A screening method was explored for simultaneous analysis of 43 cephalosporins residues and their metabolites from four generations in shrimp utilizing a novel fully untargeted data acquisition method. Four generations of cephalosporins residues and their metabolites are extracted with online extraction simultaneously. Ultrahigh-performance liquid chromatography coupled to quadrupole-orbitrap high resolution mass spectrometry was used for to determine cephalosporins and their metabolites in ten shrimp species. Precision in terms of relative standard deviation (RSD) was under 6.3% for all compounds, and the extraction recoveries ranged from 85% to 108%. The detection capabilities ranged from 0.011 to 0.223 mg kg−1. The fully untargeted vDIA acquisition way improves both selectivity and sensitivity for the compounds at trace levels, which is beneficial for screening performance.

Untargeted serum metabonomics study of psoriasis vulgaris based on ultra-performance liquid chromatography coupled to mass spectrometry.

Psoriasis is a common, chronic, systemic inflammatory skin disease, the etiology and pathogenesis is unclear. An untargeted high-throughput metabonomics method based on liquid chromatography coupled to mass spectrometry was applied to study the serum metabolic changes in psoriasis vulgaris patients, and to discover serum potential biomarkers for identification, diagnosis and exploring pathogenesis of psoriasis. The serum metabolic profiles from 150 subjects (75 psoriasis patients and 75 healthy controls) were acquired, the raw spectrometric data were processed by multivariate statistical analysis, and 44 potential biomarkers were screened out and identified. The potential biomarkers were mainly involved in glycerophospholipid metabolism, sphingolipid metabolism, arachidonic acid metabolism, bile acid biosynthesis, indicated the pathogenesis of psoriasis may be related to the disturbed metabolic pathways.

A HILIC-UHPLC-MS/MS untargeted urinary metabonomics combined with quantitative analysis of five polar biomarkers on osteoporosis rats after oral administration of Gushudan.

A HILIC-UHPLC-MS/MS untargeted urinary metabonomic method combined with quantitative analysis of five potential polar biomarkers in rat urine was developed and validated, to further understand the anti-osteoporosis effect of Gushudan(GSD) and its mechanism on prednisolone-induced osteoporosis(OP) rats in this study. The metabolites were separated and identified on Waters BEH HILIC (2.1mm×100mm, 1.7μm) column using the Waters ACQUITY™ ultra performance liquid chromatography system (Waters Corporation, Milford, USA) coupled with a Micromass Quattro Micro™ API mass spectrometer (Waters Corp, Milford, MA, USA). Principal component analysis (PCA) was used to identify potential biomarkers. Primary potential polar biomarkers including creatinine, taurine, betaine, hypoxanthine and cytosine, which were related to energy metabolism, lipid metabolism and amino acid metabolism, were found in the untargeted metabonomic research. Moreover, these targeted biomarkers were further separated and quantified in multiple-reaction monitoring (MRM) with positive ionization mode, using tinidazole as internal standard (I.S.). Good linearities (r>0.99) were obtained for all the analytes with the low limit of quantification from 1.00 to 12.8μg/mL. The relative standard deviation (RSD) of the intra-day and inter-day precisions were within 15.0% and the accuracy ranged from -14.3% to 13.5%. The recovery was more than 85.0%. And the validated method was successfully applied to investigate the urine samples of the control group, prednisolone-induced osteoporosis model group and Gushudan-treatment group in rats. Compared to the control group, the level of creatinine, taurine, betaine, hypoxanthine and cytosine in the model group revealed a significant decrease trend (p<0.05), while the Gushudan-treatment group showed no statistically differences by an independent sample t-test. This paper provided a better understanding of the therapeutic effect and mechanism of GSD on prednisolone-induced osteoporosis rats.

P3-099 - Metabolomic profiling in blood of pancreatic cancer patients

Pancreatic cancer has a poor prognosis. One of the reasons is that it is difficult to diagnose early. There has been abundant evidence that malignant cells rewire their metabolism to survive and proliferate. This study examined differentia of metabolomic profiling between patients with advanced pancreatic cancers and volunteers without ones. Ten patients and 10 volunteers who aged 20 years or older were enrolled between May and December 2015. The patients had been confirmed as pancreatic ductal adenocarcinoma cytologically or histologically. The volunteers were those who had examined abdomen CT during their health screenings and had no evidence suggesting pancreatic cancer. Blood plasma samples were derivatized and analyzed by gas chromatography mass spectrometry (GC-MS). GC-MS (Agilent 5975C MSD + 7890A GC) data produced by untargeted method were analyzed by statistical methods including principal Component Analyses (PCA). Lactic acid showed significantly high concentration with fold change of 3.23 in patients group (P ≤ 0.0001). 1,5-Anhydroglucitol was decreased with fold change of 2.94 in patients group (P ≤ 0.0001). This study suggest that pancreatic cancer patients could have different metabolomic profiling from normal population. Further investigation is required for the development of early diagnosis as well exploration of potentially therapeutic target.

Causal Genetic Variation Underlying Metabolome Differences.

An ongoing challenge in biology is to predict the phenotypes of individuals from their genotypes. Genetic variants that cause disease often change an individual's total metabolite profile, or metabolome. In light of our extensive knowledge of metabolic pathways, genetic variants that alter the metabolome may help predict novel phenotypes. To link genetic variants to changes in the metabolome, we studied natural variation in the yeast Saccharomyces cerevisiae We used an untargeted mass spectrometry method to identify dozens of metabolite Quantitative Trait Loci (mQTL), genomic regions containing genetic variation that control differences in metabolite levels between individuals. We mapped differences in urea cycle metabolites to genetic variation in specific genes known to regulate amino acid biosynthesis. Our functional assays reveal that genetic variation in two genes, AUA1 and ARG81, cause the differences in the abundance of several urea cycle metabolites. Based on knowledge of the urea cycle, we predicted and then validated a new phenotype: sensitivity to a particular class of amino acid isomers. Our results are a proof-of-concept that untargeted mass spectrometry can reveal links between natural genetic variants and metabolome diversity. The interpretability of our results demonstrates the promise of using genetic variants underlying natural differences in the metabolome to predict novel phenotypes from genotype.

New targeted approaches for the quantification of data-independent acquisition mass spectrometry.

The use of data-independent acquisition (DIA) approaches for the reproducible and precise quantification of complex protein samples has increased in the last years. The protein information arising from DIA analysis is stored in digital protein maps (DIA maps) that can be interrogated in a targeted way by using ad hoc or publically available peptide spectral libraries generated on the same sample species as for the generation of the DIA maps. The restricted availability of certain difficult-to-obtain human tissues (i.e., brain) together with the caveats of using spectral libraries generated under variable experimental conditions limits the potential of DIA. Therefore, DIA workflows would benefit from high-quality and extended spectral libraries that could be generated without the need of using valuable samples for library production. We describe here two new targeted approaches, using either classical data-dependent acquisition repositories (not specifically built for DIA) or ad hoc mouse spectral libraries, which enable the profiling of human brain DIA data set. The comparison of our results to both the most extended publically available human spectral library and to a state-of-the-art untargeted method supports the use of these new strategies to improve future DIA profiling efforts.