The structural elucidation of small molecules using mass spectrometry plays an important role in ... more The structural elucidation of small molecules using mass spectrometry plays an important role in modern life sciences and bioanalytical approaches. This review covers different soft and hard ionization techniques and figures of merit for modern mass spectrometers, such as mass resolving power, mass accuracy, isotopic abundance accuracy, accurate mass multiple-stage MS(n) capability, as well as hybrid mass spectrometric and orthogonal chromatographic approaches. The latter part discusses mass spectral data handling strategies, which includes background and noise subtraction, adduct formation and detection, charge state determination, accurate mass measurements, elemental composition determinations, and complex data-dependent setups with ion maps and ion trees. The importance of mass spectral library search algorithms for tandem mass spectra and multiple-stage MS(n) mass spectra as well as mass spectral tree libraries that combine multiple-stage mass spectra are outlined. The successi...
Methods in molecular biology (Clifton, N.J.), 2007
Metabolite profiling has been established as a multiparallel strategy for relative quantification... more Metabolite profiling has been established as a multiparallel strategy for relative quantification of a mixture of compounds or compound classes using chromatography and universal detection technologies (gas chromatography-mass spectrometry [GC-MS], liquid chromatography-MS). Despite its origens dating back to the late 1960s, it was only in the 1980s that its use was acknowledged to diagnose metabolic disorders in men, especially for rapid screening of inborn errors. Even faster electrospray ionization-MS/MS screening methods replaced longish chromatographic methods, and method development had stopped despite its potential use for other, less imminent diseases such as likelihood assessments of type II diabetes mellitus or cardiovascular risk factor evaluation. In addition to its diagnostic use, profiling blood samples can be employed to investigate specific biochemical responses. The broader scope of analysis outweighs the disadvantages by taking compromises in method development and...
Metabolomic studies are targeted at identifying and quantifying all metabolites in a given biolog... more Metabolomic studies are targeted at identifying and quantifying all metabolites in a given biological context. Among the tools used for metabolomic research, mass spectrometry is one of the most powerful tools. However, metabolomics by mass spectrometry always reveals a high number of unknown compounds which complicate in depth mechanistic or biochemical understanding. In principle, mass spectrometry can be utilized within strategies of de novo structure elucidation of small molecules, starting with the computation of the elemental composition of an unknown metabolite using accurate masses with errors <5 ppm (parts per million). However even with very high mass accuracy (<1 ppm) many chemically possible formulae are obtained in higher mass regions. In automatic routines an additional orthogonal filter therefore needs to be applied in order to reduce the number of potential elemental compositions. This report demonstrates the necessity of isotope abundance information by mathem...
Introduction The interpretation of the large number of unknown MS/MS spectra from metabolomics ex... more Introduction The interpretation of the large number of unknown MS/MS spectra from metabolomics experiments is a bottleneck in discovery-driven research. When exhaustive reference libraries are available, for example for electron ionization spectra, known structures can be easily annotated. However, metabolomic ESI MS/MS spectral libraries are still relatively small and are often not freely available. For this reason, usually fewer than 100 compounds are identified in UPLC-ESI-MS/MS experiments, despite of detecting more than 5,000 MS/MS spectra. To avoid the time-consuming manual identification when reference spectra are not available, we developed a Java-based program to use the mass spectral features such as neutral losses and m/z differences between product ions for substructure annotations. Methods Reported neutral losses and their corresponding substructures were collected from the literature. A Java program was coded to search for the mass spectral features from MS/MS spectra ...
Picture source: Fiehnlab GC-TOF-MS operated in EI mode unit resolution, low mass accuracy MS, hig... more Picture source: Fiehnlab GC-TOF-MS operated in EI mode unit resolution, low mass accuracy MS, high scan speed MS high resolution GCxGC operation LECO Pegasus IV GC-TOF-MS operated in CI mode high resolution, high mass accuracy, accurate isotopic abundances Waters GCT Premier GC-MS quadrupole operated in EI mode unit resolution, low mass accuracy MS, high scan speed MS Agilent 5975 XL 3
Introduction GC-TOF or LC-MS unbiased surveys of biological samples yield hundreds of resolved pe... more Introduction GC-TOF or LC-MS unbiased surveys of biological samples yield hundreds of resolved peaks per chromatogram. Statistical significant differences between semi-quantitative peak intensities can be routinely assigned to the classes of study designs. Metabolite peaks are then often called 'putative biomarkers' which must be validated in subsequent studies and confirmed to be specific for a diagnostic case. Consequently, the biomarker peaks must be unambiguously re-detectable over months in subsequent studies. This can best be performed by establishing standardized mass spectrometric metabolome databases. Secondly, valid biomarkers require a clear route to annotation of novel compounds to be implemented in routine clinical screens. Thirdly, interpretation of differential regulation of the identified metabolites should be guided by biochemical mapping to be of biomedical relevance.
SUMMARY: Metabolomic publications and databases use different database identifiers or even trivia... more SUMMARY: Metabolomic publications and databases use different database identifiers or even trivial names which disable queries across databases or between studies. The best way to annotate metabolites is by chemical structures, encoded by the International Chemical Identifier code (InChI) or InChIKey. We have implemented a web-based Chemical Translation Service that performs batch conversions of the most common compound identifiers, including
Systematic analysis and interpretation of the large number of tandem mass spectra (MS/MS) obtaine... more Systematic analysis and interpretation of the large number of tandem mass spectra (MS/MS) obtained in metabolomics experiments is a bottleneck in discovery-driven research. MS/MS mass spectral libraries are small compared to all known small molecule structures and are often not freely available. MS2Analyzer was therefore developed to enable user-defined searches of thousands of spectra for mass spectral features such as neutral losses, m/z differences, and product and precursor ions from MS/MS spectra in MSP/MGF files. The software is freely available at http://fiehnlab.ucdavis.edu/projects/MS2Analyzer/ . As the reference query set, 147 literature-reported neutral losses and their corresponding substructures were collected. This set was tested for accuracy of linking neutral loss analysis to substructure annotations using 19 329 accurate mass tandem mass spectra of structurally known compounds from the NIST11 MS/MS library. Validation studies showed that 92.1 ± 6.4% of 13 typical ne...
Bitterfeld (Germany) was a major site of chemical production in the former German Democratic Repu... more Bitterfeld (Germany) was a major site of chemical production in the former German Democratic Republic with chloralkali electrolysis as the basic process. Effluents were dumped via the creek Spittelwasser into the rivers Mulde and Elbe. Despite the fact that the chloralkali industry is known as a possible source of polychlorinated naphthalenes (PCNs), to date no data about PCN pollution in the region of Bitterfeld and downstream regions are available. Therefore, sediments of the creek Spittelwasser were isomer-specifically analysed for penta-, hexa-and heptachlorinated naphthalenes using GC/MS. Concentrations of 880, 543 and 1120 ng/g dry weight were found, respectively. The isomer pattern suggests chloralkali industry as the major source of PCN contamination. Because of their toxicological relevance we suggest to include PCNs into monitoring and risk assessment programs of the rivers Mulde and Elbe downstream of Bitterfeld.
The Metabolomics Standards Initiative (MSI) has recently released documents describing minimum pa... more The Metabolomics Standards Initiative (MSI) has recently released documents describing minimum parameters for reporting metabolomics experiments, in order to validate metabolomic studies and to facilitate data exchange. The reporting parameters encompassed by MSI include the biological study design, sample preparation, data acquisition, data processing, data analysis and interpretation relative to the biological hypotheses being evaluated. Herein we exemplify how such metadata can be reported by using a small case study -the metabolite profiling by GC-TOF mass spectrometry of Arabidopsis thaliana leaves from a knockout allele of the gene At1g08510 in the Wassilewskija ecotype. Pitfalls in quality control are highlighted that can invalidate results even if MSI reporting standards are fulfilled, including reliable compound identification and integration of unknown metabolites. Standardized data processing methods are proposed for consistent data storage and dissemination via databases.
Many metabolomic applications use gas chromatography/mass spectrometry (GC/MS) under standard 70 ... more Many metabolomic applications use gas chromatography/mass spectrometry (GC/MS) under standard 70 eV electron ionization (EI) parameters. However, the abundance of molecular ions is often extremely low, impeding the calculation of elemental compositions for the identification of unknown compounds. On changing the beam-steering voltage of the ion source, the relative abundances of molecular ions at 70 eV EI were increased up to ten-fold for alkanes, fatty acid methyl esters and trimethylsilylated metabolites, concomitant with 2-fold absolute increases in ion intensities. We have compared the abundance, mass accuracy and isotope ratio accuracy of molecular species in EI with those in chemical ionization (CI) with methane as reagent gas under high-mass tuning. Thirty-three peaks of a diverse set of trimethylsilylated metabolites were analyzed in triplicate, resulting in 342 ion species ([MRH] R , [M-CH 3 ] R for CI and [M] R. , [M-CH 3 ] þ. for EI). On average, CI yielded 8-fold more intense molecular species than EI. Using internal recalibration, average mass errors of 1.8 AE 1.6 mm/z units and isotope ratio errors of 2.3 AE 2.0% (Aþ1/A ratio) and 1.7 AE 1.8% (Aþ2/A ratio) were obtained. When constraining lists of calculated elemental compositions by chemical and heuristic rules using the Seven Golden Rules algorithm and PubChem queries, the correct formula was retrieved as top hit in 60% of the cases and within the top-3 hits in 80% of the cases.
Modern chemistry laboratories operate with a wide range of software applications under different ... more Modern chemistry laboratories operate with a wide range of software applications under different operating systems, such as Windows, LINUX or Mac OS X. Instead of installing software on different computers it is possible to install those applications on a single computer using Virtual Machine software. Software platform virtualization allows a single guest operating system to execute multiple other operating systems on the same computer. We apply and discuss the use of virtual machines in chemistry research and teaching laboratories.
Background: Apart from genetic alterations, development and progression of colorectal cancer has ... more Background: Apart from genetic alterations, development and progression of colorectal cancer has been linked to influences from nutritional intake, hyperalimentation, and cellular metabolic changes that may be the basis for new diagnostic and therapeutic approaches. However, in contrast to genomics and proteomics, comprehensive metabolomic investigations of alterations in malignant tumors have rarely been conducted.
The structural elucidation of small molecules using mass spectrometry plays an important role in ... more The structural elucidation of small molecules using mass spectrometry plays an important role in modern life sciences and bioanalytical approaches. This review covers different soft and hard ionization techniques and figures of merit for modern mass spectrometers, such as mass resolving power, mass accuracy, isotopic abundance accuracy, accurate mass multiple-stage MS(n) capability, as well as hybrid mass spectrometric and orthogonal chromatographic approaches. The latter part discusses mass spectral data handling strategies, which includes background and noise subtraction, adduct formation and detection, charge state determination, accurate mass measurements, elemental composition determinations, and complex data-dependent setups with ion maps and ion trees. The importance of mass spectral library search algorithms for tandem mass spectra and multiple-stage MS(n) mass spectra as well as mass spectral tree libraries that combine multiple-stage mass spectra are outlined. The successi...
Methods in molecular biology (Clifton, N.J.), 2007
Metabolite profiling has been established as a multiparallel strategy for relative quantification... more Metabolite profiling has been established as a multiparallel strategy for relative quantification of a mixture of compounds or compound classes using chromatography and universal detection technologies (gas chromatography-mass spectrometry [GC-MS], liquid chromatography-MS). Despite its origens dating back to the late 1960s, it was only in the 1980s that its use was acknowledged to diagnose metabolic disorders in men, especially for rapid screening of inborn errors. Even faster electrospray ionization-MS/MS screening methods replaced longish chromatographic methods, and method development had stopped despite its potential use for other, less imminent diseases such as likelihood assessments of type II diabetes mellitus or cardiovascular risk factor evaluation. In addition to its diagnostic use, profiling blood samples can be employed to investigate specific biochemical responses. The broader scope of analysis outweighs the disadvantages by taking compromises in method development and...
Metabolomic studies are targeted at identifying and quantifying all metabolites in a given biolog... more Metabolomic studies are targeted at identifying and quantifying all metabolites in a given biological context. Among the tools used for metabolomic research, mass spectrometry is one of the most powerful tools. However, metabolomics by mass spectrometry always reveals a high number of unknown compounds which complicate in depth mechanistic or biochemical understanding. In principle, mass spectrometry can be utilized within strategies of de novo structure elucidation of small molecules, starting with the computation of the elemental composition of an unknown metabolite using accurate masses with errors <5 ppm (parts per million). However even with very high mass accuracy (<1 ppm) many chemically possible formulae are obtained in higher mass regions. In automatic routines an additional orthogonal filter therefore needs to be applied in order to reduce the number of potential elemental compositions. This report demonstrates the necessity of isotope abundance information by mathem...
Introduction The interpretation of the large number of unknown MS/MS spectra from metabolomics ex... more Introduction The interpretation of the large number of unknown MS/MS spectra from metabolomics experiments is a bottleneck in discovery-driven research. When exhaustive reference libraries are available, for example for electron ionization spectra, known structures can be easily annotated. However, metabolomic ESI MS/MS spectral libraries are still relatively small and are often not freely available. For this reason, usually fewer than 100 compounds are identified in UPLC-ESI-MS/MS experiments, despite of detecting more than 5,000 MS/MS spectra. To avoid the time-consuming manual identification when reference spectra are not available, we developed a Java-based program to use the mass spectral features such as neutral losses and m/z differences between product ions for substructure annotations. Methods Reported neutral losses and their corresponding substructures were collected from the literature. A Java program was coded to search for the mass spectral features from MS/MS spectra ...
Picture source: Fiehnlab GC-TOF-MS operated in EI mode unit resolution, low mass accuracy MS, hig... more Picture source: Fiehnlab GC-TOF-MS operated in EI mode unit resolution, low mass accuracy MS, high scan speed MS high resolution GCxGC operation LECO Pegasus IV GC-TOF-MS operated in CI mode high resolution, high mass accuracy, accurate isotopic abundances Waters GCT Premier GC-MS quadrupole operated in EI mode unit resolution, low mass accuracy MS, high scan speed MS Agilent 5975 XL 3
Introduction GC-TOF or LC-MS unbiased surveys of biological samples yield hundreds of resolved pe... more Introduction GC-TOF or LC-MS unbiased surveys of biological samples yield hundreds of resolved peaks per chromatogram. Statistical significant differences between semi-quantitative peak intensities can be routinely assigned to the classes of study designs. Metabolite peaks are then often called 'putative biomarkers' which must be validated in subsequent studies and confirmed to be specific for a diagnostic case. Consequently, the biomarker peaks must be unambiguously re-detectable over months in subsequent studies. This can best be performed by establishing standardized mass spectrometric metabolome databases. Secondly, valid biomarkers require a clear route to annotation of novel compounds to be implemented in routine clinical screens. Thirdly, interpretation of differential regulation of the identified metabolites should be guided by biochemical mapping to be of biomedical relevance.
SUMMARY: Metabolomic publications and databases use different database identifiers or even trivia... more SUMMARY: Metabolomic publications and databases use different database identifiers or even trivial names which disable queries across databases or between studies. The best way to annotate metabolites is by chemical structures, encoded by the International Chemical Identifier code (InChI) or InChIKey. We have implemented a web-based Chemical Translation Service that performs batch conversions of the most common compound identifiers, including
Systematic analysis and interpretation of the large number of tandem mass spectra (MS/MS) obtaine... more Systematic analysis and interpretation of the large number of tandem mass spectra (MS/MS) obtained in metabolomics experiments is a bottleneck in discovery-driven research. MS/MS mass spectral libraries are small compared to all known small molecule structures and are often not freely available. MS2Analyzer was therefore developed to enable user-defined searches of thousands of spectra for mass spectral features such as neutral losses, m/z differences, and product and precursor ions from MS/MS spectra in MSP/MGF files. The software is freely available at http://fiehnlab.ucdavis.edu/projects/MS2Analyzer/ . As the reference query set, 147 literature-reported neutral losses and their corresponding substructures were collected. This set was tested for accuracy of linking neutral loss analysis to substructure annotations using 19 329 accurate mass tandem mass spectra of structurally known compounds from the NIST11 MS/MS library. Validation studies showed that 92.1 ± 6.4% of 13 typical ne...
Bitterfeld (Germany) was a major site of chemical production in the former German Democratic Repu... more Bitterfeld (Germany) was a major site of chemical production in the former German Democratic Republic with chloralkali electrolysis as the basic process. Effluents were dumped via the creek Spittelwasser into the rivers Mulde and Elbe. Despite the fact that the chloralkali industry is known as a possible source of polychlorinated naphthalenes (PCNs), to date no data about PCN pollution in the region of Bitterfeld and downstream regions are available. Therefore, sediments of the creek Spittelwasser were isomer-specifically analysed for penta-, hexa-and heptachlorinated naphthalenes using GC/MS. Concentrations of 880, 543 and 1120 ng/g dry weight were found, respectively. The isomer pattern suggests chloralkali industry as the major source of PCN contamination. Because of their toxicological relevance we suggest to include PCNs into monitoring and risk assessment programs of the rivers Mulde and Elbe downstream of Bitterfeld.
The Metabolomics Standards Initiative (MSI) has recently released documents describing minimum pa... more The Metabolomics Standards Initiative (MSI) has recently released documents describing minimum parameters for reporting metabolomics experiments, in order to validate metabolomic studies and to facilitate data exchange. The reporting parameters encompassed by MSI include the biological study design, sample preparation, data acquisition, data processing, data analysis and interpretation relative to the biological hypotheses being evaluated. Herein we exemplify how such metadata can be reported by using a small case study -the metabolite profiling by GC-TOF mass spectrometry of Arabidopsis thaliana leaves from a knockout allele of the gene At1g08510 in the Wassilewskija ecotype. Pitfalls in quality control are highlighted that can invalidate results even if MSI reporting standards are fulfilled, including reliable compound identification and integration of unknown metabolites. Standardized data processing methods are proposed for consistent data storage and dissemination via databases.
Many metabolomic applications use gas chromatography/mass spectrometry (GC/MS) under standard 70 ... more Many metabolomic applications use gas chromatography/mass spectrometry (GC/MS) under standard 70 eV electron ionization (EI) parameters. However, the abundance of molecular ions is often extremely low, impeding the calculation of elemental compositions for the identification of unknown compounds. On changing the beam-steering voltage of the ion source, the relative abundances of molecular ions at 70 eV EI were increased up to ten-fold for alkanes, fatty acid methyl esters and trimethylsilylated metabolites, concomitant with 2-fold absolute increases in ion intensities. We have compared the abundance, mass accuracy and isotope ratio accuracy of molecular species in EI with those in chemical ionization (CI) with methane as reagent gas under high-mass tuning. Thirty-three peaks of a diverse set of trimethylsilylated metabolites were analyzed in triplicate, resulting in 342 ion species ([MRH] R , [M-CH 3 ] R for CI and [M] R. , [M-CH 3 ] þ. for EI). On average, CI yielded 8-fold more intense molecular species than EI. Using internal recalibration, average mass errors of 1.8 AE 1.6 mm/z units and isotope ratio errors of 2.3 AE 2.0% (Aþ1/A ratio) and 1.7 AE 1.8% (Aþ2/A ratio) were obtained. When constraining lists of calculated elemental compositions by chemical and heuristic rules using the Seven Golden Rules algorithm and PubChem queries, the correct formula was retrieved as top hit in 60% of the cases and within the top-3 hits in 80% of the cases.
Modern chemistry laboratories operate with a wide range of software applications under different ... more Modern chemistry laboratories operate with a wide range of software applications under different operating systems, such as Windows, LINUX or Mac OS X. Instead of installing software on different computers it is possible to install those applications on a single computer using Virtual Machine software. Software platform virtualization allows a single guest operating system to execute multiple other operating systems on the same computer. We apply and discuss the use of virtual machines in chemistry research and teaching laboratories.
Background: Apart from genetic alterations, development and progression of colorectal cancer has ... more Background: Apart from genetic alterations, development and progression of colorectal cancer has been linked to influences from nutritional intake, hyperalimentation, and cellular metabolic changes that may be the basis for new diagnostic and therapeutic approaches. However, in contrast to genomics and proteomics, comprehensive metabolomic investigations of alterations in malignant tumors have rarely been conducted.
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Papers by Tobias Kind