Project description:Non-targeted analysis (NTA) using high-resolution mass spectrometry has enabled the detection and identification of unknown and unexpected compounds of interest in a wide range of sample matrices. Despite these benefits of NTA methods, standardized procedures do not yet exist for assessing performance, limiting stakeholders' abilities to suitably interpret and utilize NTA results. Herein, we first summarize existing performance assessment metrics for targeted analyses to provide context and clarify terminology that may be shared between targeted and NTA methods (e.g., terms such as accuracy, precision, sensitivity, and selectivity). We then discuss promising approaches for assessing NTA method performance, listing strengths and key caveats for each approach, and highlighting areas in need of further development. To structure the discussion, we define three types of NTA study objectives: sample classification, chemical identification, and chemical quantitation. Qualitative study performance (i.e., focusing on sample classification and/or chemical identification) can be assessed using the traditional confusion matrix, with some challenges and limitations. Quantitative study performance can be assessed using estimation procedures developed for targeted methods with consideration for additional sources of uncontrolled experimental error. This article is intended to stimulate discussion and further efforts to develop and improve procedures for assessing NTA method performance. Ultimately, improved performance assessments will enable accurate communication and effective utilization of NTA results by stakeholders.
Project description:With the increasing availability of high-resolution mass spectrometers, suspect screening and non-targeted analysis are becoming popular compound identification tools for environmental researchers. Samples of interest often contain a large (unknown) number of chemicals spanning the detectable mass range of the instrument. In an effort to separate these chemicals prior to injection into the mass spectrometer, a chromatography method is often utilized. There are numerous types of gas and liquid chromatographs that can be coupled to commercially available mass spectrometers. Depending on the type of instrument used for analysis, the researcher is likely to observe a different subset of compounds based on the amenability of those chemicals to the selected experimental techniques and equipment. It would be advantageous if this subset of chemicals could be predicted prior to conducting the experiment, in order to minimize potential false-positive and false-negative identifications. In this work, we utilize experimental datasets to predict the amenability of chemical compounds to detection with liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS). The assembled dataset totals 5517 unique chemicals either explicitly detected or not detected with LC-ESI-MS. The resulting detected/not-detected matrix has been modeled using specific molecular descriptors to predict which chemicals are amenable to LC-ESI-MS, and to which form(s) of ionization. Random forest models, including a measure of the applicability domain of the model for both positive and negative modes of the electrospray ionization source, were successfully developed. The outcome of this work will help to inform future suspect screening and non-targeted analyses of chemicals by better defining the potential LC-ESI-MS detectable chemical landscape of interest.
Project description:Hemoglobinopathies are the most common inherited disorders worldwide. Accurate analysis of hemoglobin variants is critical for diagnosis of hemoglobinopathies. Although high-performance liquid chromatography and capillary zone electrophoresis are widely used as screening tools, they possess inherent ambiguities that often preclude accurate detection of hemoglobin variants. Our goal was to develop and optimize a sensitive and specific mass spectrometry-based assay for screening and diagnosis of hemoglobinopathies. A catalog of canonical globin-chain specific peptides as well as mutant peptides corresponding to common hemoglobin variants was generated and their corresponding “heavy” synthetic peptide versions were used as internal standards for quantification and calculation of globin chain ratios. Targeted mass spectrometry analysis was performed by coupling liquid chromatography to a triple quadrupole mass spectrometer, which is the commonest mass spectrometer employed in clinical diagnostics. Dried blood spots from a cohort of 716 individuals (including 211 patients with hemoglobinopathy) were analyzed. The α:β-globin ratios showed a significant difference between normal and β-thalassemia patients, particularly when the disease was homozygous or admixed with structural variants (compound heterozygous). The method presented here permits identification of variants in their homozygous, heterozygous or compound heterozygous states. The intra- and inter-assay precision CV were both <20%. We envision that such mass spectrometry-based assays could be employed as first-line screening assay for hemoglobin variants including sickle cell disease as well as thalassemias.
Project description:Fabry disease (FD) is an X-linked lysosomal storage disorder where impaired α-galactosidase A enzyme activity leads to the intracellular accumulation of undegraded glycosphingolipids, including globotriaosylsphingosine (lyso-Gb3) and related analogues. Lyso-Gb3 and related analogues are useful biomarkers for screening and should be routinely monitored for longitudinal patient evaluation. In recent years, a growing interest has emerged in the analysis of FD biomarkers in dried blood spots (DBSs), considering the several advantages compared to venipuncture as a technique for collecting whole-blood specimens. The focus of this study was to devise and validate a UHPLC-MS/MS method for the analysis of lyso-Gb3 and related analogues in DBSs to facilitate sample collection and shipment to reference laboratories. The assay was devised in conventional DBS collection cards and in Capitainer®B blood collection devices using both capillary and venous blood specimens from 12 healthy controls and 20 patients affected with FD. The measured biomarker concentrations were similar in capillary and venous blood specimens. The hematocrit (Hct) did not affect the correlation between plasma and DBS measurements in our cohort (Hct range: 34.3-52.2%). This UHPLC-MS/MS method using DBS would facilitate high-risk screening and the follow-up and monitoring of patients affected with FD.
Project description:A fully non-targeted analytical workflow for the investigation of a riverbank filtration site located at the river Danube has been developed and applied. Variations of compound intensities at different sampling locations of the riverbank filtration site and, for a single production well, over a monitoring period of one year have been investigated using liquid chromatography combined with time-of-flight-mass spectrometry followed by evaluation via non-targeted data analysis. Internal standardization and appropriate quality control strategies have been implemented into the workflow for reduction of possible methodological biases influencing data interpretation. Emphasis was placed on the assessment of different blank elimination steps and the final blank elimination strategy is reported. The spatial study of the selected riverbank filtration site revealed a homogenous composition of the filtered water sampled at 11 different locations across the 32,000 m2 site, except for one sampling location in a zone of the aquifer, which was only weakly connected to the well field in terms of hydrogeological conditions. The examination of time-dependent changes of the composition of surface and groundwater obtained at the riverbank filtration system revealed that the non-targeted workflow is fit-for-purpose regarding the assessment the stability of filtration efficiency and compound residence time in the riverbank filtration compartment. In total, 677 compounds were selected for the investigation of the time-dependent variations of the filtration process. Analysis of the signal intensities of these compounds revealed that the riverbank filtration is significantly reducing the intensity and number of compounds present in surface water over a wide polarity range. In addition, the method enabled the determination of compound residence times in the riverbank filtration system ranging from 5 to 7 days.
Project description:Aldosterone-producing adenomas (APAs) have different steroid profiles in serum, depending on the causative genetic mutation. Ion mobility is a separation technique for gas-phase ions based on their m/z values, shapes, and sizes. Human serum (100 µL) was purified by liquid-liquid extraction using tert-butyl methyl ether/ethyl acetate at 1/1 (v/v) and mixed with deuterium-labeled steroids as the internal standard. The separated supernatant was dried, re-dissolved in water containing 20% methanol, and injected into a liquid chromatography-ion mobility-mass spectrometer (LC/IM/MS). We established a highly sensitive assay system by separating 20 steroids based on their retention time, m/z value, and drift time. Twenty steroids were measured in the serum of patients with primary aldosteronism, essential hypertension, and healthy subjects and were clearly classified using principal component analysis. This method was also able to detect phosphatidylcholine and phosphatidylethanolamine, which were not targeted. LC/IM/MS has a high selectivity for known compounds and has the potential to provide information on unknown compounds. This analytical method has the potential to elucidate the pathogenesis of APA and identify unknown steroids that could serve as biomarkers for APA with different genetic mutations.
Project description:Targeted proteomics recently proved to be a technique for the detection and absolute quantification of proteins not easily accessible to classical bottom-up approaches. Due to this, it has been considered as a high fidelity tool to detect potential warfare agents in wide spread kinds of biological and environmental matrices. Clostridium perfringens toxins are considered to be potential biological weapons, especially the epsilon toxin which belongs to a group of the most powerful bacterial toxins. Here, the development of a target mass spectrometry method for the detection of C. perfringens protein toxins (alpha, beta, beta2, epsilon, iota) is described. A high-resolution mass spectrometer with a quadrupole-Orbitrap system operating in target acquisition mode (parallel reaction monitoring) was utilized. Because of the lack of commercial protein toxin standards recombinant toxins were prepared within Escherichia coli. The analysis was performed using proteotypic peptides as the target compounds together with their isotopically labeled synthetic analogues as internal standards. Calibration curves were calculated for each peptide in concentrations ranging from 0.635 to 1101 fmol/μL. Limits of detection and quantification were determined for each peptide in blank matrices.
Project description:Accumulation of propionylcarnitine (C3) in neonatal dried blood spots (DBS) is indicative of inborn errors of propionate metabolism including propionic acidemia (PA), methylmalonic aciduria (MMA), and cobalamin (Cbl) metabolic defects. Concentrations of C3 in affected newborns overlap with healthy individuals rendering this marker neither specific nor sensitive. While a conservative C3 cutoff together with relevant acylcarnitines ratios improve screening sensitivity, existing mass spectrometric methods in newborn screening laboratories are inadequate at improving testing specificity. Therefore, using the original screening DBS, we sought to measure 2-methylcitric acid (MCA), a pathognomonic hallmark of C3 disorders to decrease the false positive rate and improve the positive predictive value of C3 disorders. MCA was derivatized with 4-[2-(N,N-dimethylamino)ethylaminosulfonyl]-7-(2-aminoethylamino)-2,1,3-benzoxadiazole (DAABD-AE). No separate extraction step was required and derivatization was performed directly using a 3.2-mm disc of DBS as a sample (65°C for 45 min). The reaction mixture was analyzed by liquid chromatography tandem mass spectrometry. MCA was well separated and eluted at 2.3 min with a total run time of 7 min. The median and (range) of MCA of 0.06 ?mol/L (0-0.63) were in excellent agreement with the literature. The method was applied retrospectively on DBS samples from established patients with PA, MMA, Cbl C, Cbl F, maternal vitamin B12 deficiency (n?=?20) and controls (n?=?337). Comparison with results obtained by another method was satisfactory (n?=?252). This method will be applied as a second tier test for samples which trigger positive PA or MMA results by the primary newborn screening method.
Project description:Neonatal dried blood spots (DBS) are routinely utilized in the clinical setting as a diagnostic tool for various genetic disorders and infectious diseases. DBS allow for minimally invasive, small volume blood collection and are stored at room temperature. Neonatal whole blood and serum samples can be important in determining genetic risk factors and predicting infantile disease; however, at the present time, limited methods exist for rapidly analyzing DBS samples for their proteomic profile, years after samples have been collected. A novel method is presented for the extraction and analysis of target proteins and peptides from neonatal DBS using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Extraction parameters were optimized to achieve ideal signal intensity and resolution to obtain protein identifications. Samples were extracted from filter paper with 0.1% TFA in H2O for 72 h. The extract was subjected to enzymatic digestion, spotted on an ITO-coated glass slide, and washed in order to remove salts. Analysis of extracted blood spots from ten newborns was completed. Similarities and differences in the proteomic profile of the washed extracts are presented, herein, to verify the viability of this method for analysis of dated DBS samples. This method allows for analysis of DBS samples years after collection and can be utilized to correlate diseases or disorders manifesting later in life with potential risk factors presenting in the proteomic profile of the DBS collected at time of birth.
Project description:BackgroundThis study sought to analyze non-targeted plasma metabolites in patients with atherosclerosis (AS).MethodsThe plasma of patients with AS (the patient group) and the plasma of age-matched and gender-matched healthy individuals (the control group) at the Taihe Hospital was collected. One hundred patients were included in the study (60 in the patient group and 40 in the control group). Fasting venous plasma was collected in the morning. The metabolites in the plasma were examined by liquid chromatography-mass spectrometry (LC-MS). An unsupervised principal component analysis (PCA) was conducted to observe the overall distribution of each sample and the stability of the analysis process. Next, a supervised partial least squares-discriminant analysis (PLS-DA) and an orthogonal partial least squares-discriminant analysis (OPLS-DA) were conducted to examine the overall differences among the metabolic profiles of the groups and identify different metabolites in the groups. Pathway enrichment was analyzed using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database.ResultsIn total, 1,126 different metabolites were detected in the patient and control groups. Compared to the control group, 411 species decreased, and 715 species increased in the patient group. There were 61 different metabolites with a variable weight in the projection (VIP) >1 and a P<0.05. There were 34 types of lipid metabolites, 10 types of carbon and oxygen compounds, 8 types of organic acids and derivatives, 4 types of organoheterocyclic compounds, 3 types of nitrogen-containing organic compounds, and 2 types of nucleotides and analogs. Compared to the control group, 47 species decreased, and 14 species increased in the patient group. The following 9 metabolites had the most significant differences (|log2fold change| >1; P<0.05): 2-tetradecanone, pantothenol, all-trans-13,14-dihydroretinol, linoleoyl ethanolamide, N-oleoylethanolamine, 4-methyl-2-pentenal, Cer (d18:1/14:0), chenodeoxycholic acid glycine conjugate, and 5-acetamidovalerate. The enrichment analysis results of the 61 different metabolite pathways identified 17 metabolic pathways with significant differences (P<0.05), including the choline metabolism, lipid metabolism, autophagy, amino acid metabolism, vitamin digestion, and absorption pathways.ConclusionsThere are significant differences in non-targeted plasma metabolites between patients with AS and healthy individuals. The above-mentioned 9 most significantly different metabolites may be potential markers of AS.