Untargeted analysis of mass spectrometry data for elucidation of metabolites and function of enzymes

A Matlab-based computer program termed Discovery of General Endo- and Xenobiotics (DoGEX) was developed, which uses wavelets and morphological analysis to process liquid chromatography-mass spectrometry (LC-MS) data. The output of the program is a list of integration areas as a function of retention time and molecular mass. A feature of the computer program is spectral filtering to facilitate the detection of chromatographic peaks with a particular isotopic ratio. The program DoGEX was used to automatically select oxidation products formed from felodipine (i.e., two chlorine atoms) and bromocriptine (one bromine atom) with cytochrome P450 3A4. The recognized isotope ratio can be changed to permit a natural or artificial mixture of isotopes to be monitored for selections. This computer program can be used to analyze LC-MS data for untargeted metabolic profiling experiments, e.g., to assign endogenous functions to newly characterized cytochrome P450 enzymes. In a representative example, an incubation of testosterone, NADPH, and a 1:1 16O2/18O2 mixture yielded products with M and M+2 ions resembling bromine doublets. Another use of the program is the subtraction of one set of tR, m/z data from another, e.g., in comparisons of changes in patterns during enzyme reactions.     

Identification of di-isononyl phthalate metabolites for exposure marker discovery using in vitro/in vivo metabolism and signal mining strategy with LC-MS data

Di-isononyl phthalate esters (DINPs) are endocrine-disrupting chemicals and have replaced di(2-ethylhexyl) phthalate (DEHP) as the major plasticizer for poly(vinyl chloride) (PVC) products in recent years. Exposure marker discovery of DINPs is crucial, because of their high potential for human exposure and toxicity. Here, we propose an alternative approach for tracing signals derived from stable isotope-labeled precursors with varied labeling ratios to efficiently filter probable metabolite signals. The statistical process, which involves a signal mining algorithm with isotope tracing (SMAIT), has effectively filtered 13 probable DINP metabolite signals out of the 8867 peaks in the LC-MS data obtained from incubated stable isotope-labeled precursors with liver enzymes. Seven of the 13 probable metabolite signals were confirmed as DINP structure-related metabolites by preliminary MS/MS analyses. These 7 structure-related metabolite signals were validated as effective DINP exposure markers, using urine samples collected from DINP-administered rats without time-consuming comprehensive structure identification. We propose that the 7 identified possible DINP metabolite signals of m/z 279.1, 293.1, 305.1, 307.1, 321.1, 365.1, and 375.1 are potential markers for DINP exposure and should be investigated further. The integrated approach described here can efficiently, and systematically, filter probable metabolite signals from a complex LC-MS dataset for toxic exposure marker discovery. It is a relatively low-cost/rapid workflow for exposure marker discovery.     

Comparison of different mass spectrometric techniques combined with liquid chromatography for confirmation of pesticides in environmental water based on the use of identification points

Three mass spectrometric techniques have been used and compared for the confirmation of the presence of several pesticides that had been detected in environmental water samples by a previously reported SPE-LC-MS/MS screening method. The 2002/657/EC European Comission Decision establishes the need to obtain at least three identification points (IPs) in order to confirm organic residues and contaminants in live animals and animal products. In this paper, a similar approach has been applied for confirmation of pesticides in water samples, using triple quadrupole mass spectrometry (QqQ), time-of-flight mass spectrometry (TOF), and hybrid quadrupole time-of-flight mass spectrometry (QTOF) to achieve the required IPs. The number of IPs collected, the sensitivity, and the practical advantages and disadvantages of these techniques have been discussed. In summary, the QqQ instrument allowed the confirmation of detected pesticides even at very low concentrations (ng/L) achieving between four and five IPs when adding confirmatory transitions. The direct confirmation with a TOF instrument was only feasible for those compounds showing sufficient sensitivity, isotopic pattern, or easy in-source fragmentation. In other cases, the required IPs could be reached by adding IPs earned with this technique to those obtained from the MS/MS screening method. Finally, the use of a QTOF instrument allowed obtaining up to 20 IPs in a single run at relatively high concentrations (submicrograms per liter) as no "ion shopping" was required. Additionally, the application of TOF and QTOF techniques made it possible to detect some nontarget organic contaminants, which were not included in the screening method.     

Capillary action-supported contactless atmospheric pressure ionization for the combined sampling and mass spectrometric analysis of biomolecules

It is proposed that a short tapered capillary can be utilized as a nanoliter-volume sampling tool and sample emitter for generation of gas-phase ions in front of the mass spectrometer, without the need for using an additional electric power supply, a gas supply, or a syringe pump. A wide range of molecules can be analyzed in pure solutions and complex matrixes (cell extract, urine, and plant tissue) with no or minimum sample preparation. Singly and multiply charged ions can be detected in either positive or negative-ion mode. Because of the nanoliter-volume sampling and low spectral background, the mass detection limit for bradykinin is in the low attomole range. Other advantages include simplicity, disposability, and low cost. The putative mechanism of the ion formation in this capillary-action supported contactless spray emitter is discussed.     

Review of procedures involving separation and preconcentration for the determination of cadmium using spectrometric techniques

Spectrometric techniques for the analysis of trace cadmium have developed rapidly due to the increasing need for accurate measurements at extremely low levels of this element in diverse matrices. This review covers separation and preconcentration procedures, such as electrochemical deposition, precipitation, coprecipitation, solid phase extraction, liquid-liquid extraction (LLE) and cloud point extraction (CPE), and consider the features of the their application with several spectrometric techniques.     

Identification of bacteria using tandem mass spectrometry combined with a proteome database and statistical scoring

Detection and identification of pathogenic bacteria and their protein toxins play a crucial role in a proper response to natural or terrorist-caused outbreaks of infectious diseases. The recent availability of whole genome sequences of priority bacterial pathogens opens new diagnostic possibilities for identification of bacteria by retrieving their genomic or proteomic information. We describe a method for identification of bacteria based on tandem mass spectrometric (MS/MS) analysis of peptides derived from bacterial proteins. This method involves bacterial cell protein extraction, trypsin digestion, liquid chromatography MS/MS analysis of the resulting peptides, and a statistical scoring algorithm to rank MS/MS spectral matching results for bacterial identification. To facilitate spectral data searching, a proteome database was constructed by translating genomes of bacteria of interest with fully or partially determined sequences. In this work, a prototype database was constructed by the automated analysis of 87 publicly available, fully sequenced bacterial genomes with the GLIMMER gene finding software. MS/MS peptide spectral matching for peptide sequence assignment against this proteome database was done by SEQUEST. To gauge the relative significance of the SEQUEST-generated matching parameters for correct peptide assignment, discriminant function (DF) analysis of these parameters was applied and DF scores were used to calculate probabilities of correct MS/MS spectra assignment to peptide sequences in the database. The peptides with DF scores exceeding a threshold value determined by the probability of correct peptide assignment were accepted and matched to the bacterial proteomes represented in the database. Sequence filtering or removal of degenerate peptides matched with multiple bacteria was then performed to further improve identification. It is demonstrated that using a preset criterion with known distributions of discriminant function scores and probabilities of correct peptide sequence assignments, a test bacterium within the 87 database microorganisms can be unambiguously identified.     

Identification of uncommon plant metabolites based on calculation of elemental compositions using gas chromatography and quadrupole mass spectrometry

Unknown compounds in polar fractions of Arabidopsis thaliana crude leaf extracts were identified on the basis of calculations of elemental compositions obtained from gas chromatography/low-resolution quadrupole mass spectrometric data. Plant metabolites were methoximated and silylated prior to analysis. All known peaks were used as internal references to construct polynomial recalibration curves of from raw mass spectrometric data. Mass accuracies of 0.005 +/- 0.003 amu and isotope ratio errors of 0.5 +/- 0.3% (A + 1/A), respectively, 0.3 +/- 0.2% (A + 2/A), could be achieved. Both masses and isotope ratios were combined when the elemental compositions of unknown peaks were calculated. After calculation, compound identities were elucidated by searching metabolic databases, interpreting spectra, and, finally, by comparison with reference compounds. Sum formulas of more than 70 peaks were determined throughout single GC/MS chromatograms. Exact masses were confirmed by high-resolution mass spectrometric data. More than 15 uncommon plant metabolites were identified, some of which are novel in Arabidopsis, such as tartronate semialdehyde, citramalic acid, allothreonine, or glycolic amide.     

Derivatization using dimethylamine for tandem mass spectrometric structure analysis of enzymatically and acidically depolymerized methyl cellulose

Structure analysis of partially depolymerized methyl cellulose was performed by nanoelectrospray ionization tandem mass spectrometry (nano-ESI-MS/MS) and by matrix-assisted laser desorption/ionization tandem mass spectrometry (MALDI-MS/MS). Dimethylamine (DMA) was used for the first time as a reducing end derivatization reagent for oligosaccharides. This is an attractive reagent since it could be easily removed from the reaction mixture. Most important it also introduces a basic functional group that increased the sensitivity in both MALDI and nano-ESI. Depolymerization was made in two ways: one by the cellulose selective endoglucanase 5A from Bacillus agaradhaerens (Ba Cel5A) and the other by trifluoroacetic acid. The DMA derivatives formed both protonated and sodiated molecules in nano-ESI and MALDI. Tandem MS of protonated molecules yielded predominantly Y fragments from which the distribution of the substituents in the oligomers could be measured. Fragments obtained in tandem MS of sodiated molecules provided information regarding the positions of the substituents within the anhydroglucose units (AGUs). It was found that Ba Cel5A could cleave glucosidic bonds also if the AGU on the reducing side of the bond was fully methylated. The combination of DMA derivatization and tandem MS was demonstrated as a tool for the characterization of endoglucanase selectivity.     

Speciation and identification of organoselenium metabolites in human urine using inductively coupled plasma mass spectrometry and tandem mass spectrometry

A method for speciation and identification of organoselenium metabolites found in human urine samples using high performance liquid chromatography/inductively coupled plasma mass spectrometry (HPLC/ICP-MS) and tandem mass spectrometry (MS/MS) is described. Reversed-phase chromatographic separation was used for sample fractionation with the ICP-MS functioning as an element-selective detector, and six distinct selenium-containing species were detected in a human urine sample. Fractions were then collected and analyzed using a triple quadrupole mass spectrometer with electrospray ionization and collision-induced dissociation to obtain structural information. The first two fractions were identified specifically as selenomethionine and selenocystamine, estimated to be present at approximately 11 and 40 ppb, respectively. To the best of our knowledge, this is the first time these two metabolites have been positively identified in human urine.     

Practical approach for the identification and isomer elucidation of biomarkers detected in a metabonomic study for the discovery of individuals at risk for diabetes by integrating the chromatographic and mass spectrometric information

Sensitive and high-resolution chromatographic-driven metabonomomics studies experienced major growth with the aid of new analytical technologies and bioinformatics software packages. Hence, data collections by LC-MS and data analyses by multivariate statistical methods are by far the most straightforward steps, and the detection of biomarker candidates can easily be achieved. However, the unequivocal identification of the detected metabolite candidates, including isomer elucidation, is still a crux of current metabonomics studies. Here we present a comprehensive analytical strategy for the elucidation of the molecular structure of metabolite biomarkers detected in a metabonomics study, exemplified analyzing spot urine of a cohort of healthy, insulin sensitive subjects and clinically well characterized prediabetic, insulin resistant individuals. An integrated approach of LC-MS fingerprinting, multivariate statistic analysis, LC-MSn experiments, micro preparation, FTICR-MS, GC retention index, database search, and generation of an isotope labeled standard was applied. Overall, we could demonstrate the efficiency of our analytical approach by the unambiguous elucidation of the molecular structure of an isomeric biomarker candidate detected in a complex human biofluid. The proposed strategy is a powerful new analytical tool, which will allow the definite identification of physiologically important molecules in metabonomics studies from basic biochemistry to clinical biomarker discovery.     

Identification and measurement of illicit drugs and their metabolites in urban wastewater by liquid chromatography-tandem mass spectrometry

Residues of illicit drugs and their metabolites that are excreted by humans may flow into and through wastewater treatment plants. The aim of this study was to develop a method for the determination of cocaine, amphetamines, morphine, cannabinoids, methadone, and some of their metabolites in wastewater. Composite 24-h samples from urban treatment plants were enriched with deuterated internal standards before solid-phase extraction. High-pressure liquid chromatography tandem mass spectrometry with multiple reaction monitoring was used for quantitation. Recoveries were generally higher than 80%, and limits of quantifications were in the low nanograms-per-liter range for untreated and treated wastewater. The overall variability of the method was lower than 10% for untreated and 5% for treated wastewater. The method was applied to wastewater samples coming from two treatment plants in Italy and Switzerland. Quantification ranges were found to be 0.2-1 microg/L for cocaine and its metabolite benzoylecgonine, 80-200 ng/L for morphine, 10 ng/L for 6-acetylmorphine, 60-90 ng/L for 11-nor-9-carboxy-Delta9-tetrahydrocannabinol, 10-90 ng/L for methadone and its main metabolite 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine, and lower than 20 ng/L for amphetamines. As previously reported for cocaine, this method could be useful to estimate and monitor drug consumption in the population in real time, helping social scientists and authorities to combat drug abuse.     

Identification of phosphoserine and phosphothreonine as cysteic acid and beta-methylcysteic acid residues in peptides by tandem mass spectrometric sequencing

Tandem mass spectrometry has long been an intrinsic tool to determine phosphorylation sites in proteins. However, loss of the phosphate moiety from both phosphoserine and phosphothreonine residues in low-energy collision-induced dissociation is a common phenomenon, which makes identification of P-Ser and P-Thr residues complicated. A method for direct sequencing of the Ser and Thr phosphorylation sites by ESI tandem mass spectrometry following beta-elimination/sulfite addition to convert -HPO4 to -SO3 has been studied. Five model phosphopeptides, including three synthetic P-Ser-, P-Thr-, or P-Ser- and P-Thr-containing peptides; a protein kinases C-phosphorylated peptide; and a phosphopeptide derived from beta-casein trypsin digests were modified and then sequenced using an ESI-quadrupole ion trap mass spectrometer. Following incubation of P-Ser- or P-Thr-containing peptides with Na2SO3/NaOH, 90% P-Ser and 80% P-Thr was converted to cysteic acid and beta-methylcysteic acid, respectively, as revealed by amino acid analysis. The conversion can be carried out at 1 microM concentration of the peptide. Both cysteic acid and beta-methylcysteic acid residues in the sequence were shown to be stable and easily identifiable under general conditions for tandem mass spectrometric sequencing applicable to common peptides.     

Determination of carbamazepine and its metabolites in aqueous samples using liquid chromatography-electrospray tandem mass spectrometry

A quantitative method is described for solid-phase extraction (SPE) followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the simultaneous analysis of carbamazepine and its five metabolites, 10,11-dihydro-10,11-epoxycarbamazepine, 10,11-dihydro-10,11-dihydroxycarbamazepine, 2-hydroxycarbamazepine, 3-hydroxycarbamazepine, and 10,11-dihydro-10-hydroxycarbamazepine. An SPE procedure was used to concentrate target compounds from aqueous samples collected from sewage treatment plant (STP) wastewater and surface water. Extracts were analyzed using electrospray LC-MS/MS with time-scheduled selected reaction monitoring. The recoveries of the analytes were 83.6-102.2% from untreated sewage (influent), 90.6-103.5% from treated sewage (effluent), and 95.7-102.9% from surface water samples. The instrumental detection limits were 0.8-4.8 pg for the analytes. Matrix effects were investigated for the analytes in HPLC-grade water, surface water, and STP influent and effluent. Ion suppression increased for analytes in order of surface water to STP effluent to STP influent, but no ion suppression was observed for analytes in HPLC-grade water. The developed method was validated by analysis of environmental aqueous samples: STP influent and effluent and surface water. Carbamazepine and all five metabolites were detected in STP influent and effluent samples. Only carbamazepine and 10,11-dihydro-10,11-dihydroxycarbamazepine were detected in the surface water sample. Notably, 10,11-dihydro-10,11-dihydroxycarbamazepine was detected at approximately 3 times higher concentrations than the parent drug, carbamazepine, in all of the aqueous samples. To our knowledge, this is the first report on the simultaneous determination of carbamazepine and its metabolites in environmental samples.     

Applying in-silico retention index and mass spectra matching for identification of unknown metabolites in accurate mass GC-TOF mass spectrometry

One of the major obstacles in metabolomics is the identification of unknown metabolites. We tested constraints for reidentifying the correct structures of 29 known metabolite peaks from GCT premier accurate mass chemical ionization GC-TOF mass spectrometry data without any use of mass spectral libraries. Correct elemental formulas were retrieved within the top-3 hits for most molecular ion adducts using the "Seven Golden Rules" algorithm. An average of 514 potential structures per formula was downloaded from the PubChem chemical database and in-silico-derivatized using the ChemAxon software package. After chemical curation, Kovats retention indices (RI) were predicted for up to 747 potential structures per formula using the NIST MS group contribution algorithm and corrected for contribution of trimethylsilyl groups using the Fiehnlib RI library. When matching the range of predicted RI values against the experimentally determined peak retention, all but three incorrect formulas were excluded. For all remaining isomeric structures, accurate mass electron ionization spectra were predicted using the MassFrontier software and scored against experimental spectra. Using a mass error window of 10 ppm for fragment ions, 89% of all isomeric structures were removed and the correct structure was reported in 73% within the top-5 hits of the cases.     

Investigation of heat and mass transfer in a rarefied gas using high-frequency measurement techniques

Devices based on a high-frequency generator for investigating heat and mass transfer in a rarefied gas — solid system are described. Their fundamental characteristics are given and procedures and results of tests are presented. __________ Translated from Izmeteritel’naya Tekhnika, No. 3, pp. 49–54, March, 2006.     

Rationalization of retention and overloading behavior of basic compounds in reversed-phase HPLC using low ionic strength buffers suitable for mass spectrometric detection

The retention and overloading behavior of some basic (and acidic) compounds has been studied on different RP-HPLC columns in buffers of varying ionic strength. Anomalous retention patterns of acids and bases were found on one phase in low-pH, volatile buffers such as formic acid, favored for mass spectrometric analysis. Unusual retention compared to that in higher ionic strength phosphate buffers is attributed to the presence of positively charged sites existing on this phase at low pH. Overloading of bases as well as acids is shown to be a function of mobile-phase ionic strength. This result is a logical consequence of previous suggestions that mutual repulsion of ions held on the hydrophobic surface of the stationary phase, rather than overload of silanols, is largely responsible for overloading on pure silica RP columns. Thus, overloading occurs much more readily in low ionic strength formic acid buffers. Appreciable loss of efficiency can occur in such buffers when only 50 ng of some bases is analyzed on a standard-sized column.     

Application of atmospheric pressure ionization time-of-flight mass spectrometry coupled with liquid chromatography for the characterization of in vitro drug metabolites

Atmospheric pressure ionization time-of-flight mass spectrometry coupled with high-performance liquid chromatography was used to characterize the in vitro metabolites of glyburide. Metabolic products formed in vitro by human microsomes were separated using a C18 column with gradient elution at a flow rate of 200 microL/min without postcolumn splitting. In-source collision-induced dissociation (CID) by automated nozzle potential switching was employed to obtain both abundant protonated molecules and characteristic fragments whose accurate masses were measured simultaneously by internal mass calibration, performed by continuous postcolumn infusion of two reference standards. The mass errors were within 9 ppm for all ions measured, whose abundance was greater than 5%, relative to the most abundant isotopic "A" ion. Exact mass differences between the parent drug and metabolite(s) were determined and these values corresponded to a unique elemental composition. The elemental compositions of all metabolite fragment ions were generated based upon the known compositional elements of the protonated molecule. The structures of metabolites and their fragment ions were proposed based on the determined elemental composition and in-source CID spectra. The elemental composition and fragmentation pathways of four cyclohexyl hydroxylation metabolites and one ethylhydroxy metabolite are discussed.     

Identification of rotenone-induced modifications in alpha-synuclein using affinity pull-down and tandem mass spectrometry

Parkinson's disease is a movement disorder that results from a loss of dopaminergic neurons in the substantia nigra. The disease is characterized by mitochondrial dysfunction, oxidative stress, and the presence of "Lewy body" inclusions enriched with aggregated forms of alpha-synuclein, a presynaptic protein. Although alpha-synuclein is modified at various sites in Lewy bodies, it is unclear how sequence-specific posttranslational modifications modulate the aggregation of the protein in oxidatively stressed neurons. To begin to address this problem, we developed an affinity pull-down/mass spectrometry method to characterize the primary structure of histidine-tagged alpha-synuclein isolated from catecholaminergic neurons. Using this method, we mapped posttranslational modifications of alpha-synuclein from untreated neurons and neurons exposed to rotenone, an inhibitor of mitochondrial complex I. Various posttranslational modifications suggestive of oxidative damage or repair were identified in a region comprising a 20-residue stretch in the C-terminal part of the protein. The results indicate that alpha-synuclein is subject to discrete posttranslational modifications in neurons with impaired mitochondrial function. Our affinity pull-down/mass spectrometry method is a useful tool to examine how specific modifications of alpha-synuclein contribute to neurologic disorders such as Parkinson's disease.