Using Orbitrap mass spectrometry to assess the isotopic compositions of individual compounds in mixtures

The isotopic compositions of individual chemical species are routinely used by the geochemical, environmental, forensic, anthropological, chemical, and biomedical communities to elucidate the conditions, sources, and reaction pathways of the molecules in question. Mass spectrometric methods of measuring isotopic compositions of individual compounds generally require that analytes be pure to yield precise, accurate results, yet most applications examine materials that are mixtures of multiple components. Various methods of chemical purification, e.g., chromatography, are used to isolate analytes from mixtures prior to mass spectrometric analysis. However, these techniques take time and specialized instrumentation, both of which could potentially be obviated via the use of ultra-high-resolution mass spectrometry. Here we report on the use of Orbitrap™-based Fourier-transform mass spectrometry to perform isotope ratio measurements of single species within mixtures delivered to the mass spectrometer (MS) without prior chromatographic separation. We demonstrate that instrument biases (attributed here to space charge effects) within the Orbitrap mass analyzer can cause the measured ¹³C/¹²C ratio of a molecular ion in the presence of non-analyte-derived ‘contaminating’ species to spuriously decrease relative to the ¹³C/¹²C ratio measured for the same ion in a pure analyte. We observe that the decrease in ¹³C/¹²C is proportional to the relative concentrations of the additional ‘contaminating’ components. We then recommend several strategies by which this effect can be mediated such that accurate isotope ratios can be obtained

Using Orbitrap mass spectrometry to assess the isotopic compositions of individual compounds in mixtures

The isotopic compositions of individual chemical species are routinely used by the geochemical, environmental, forensic, anthropological, chemical, and biomedical communities to elucidate the conditions, sources, and reaction pathways of the molecules in question. Mass spectrometric methods of measuring isotopic compositions of individual compounds generally require that analytes be pure to yield precise, accurate results, yet most applications examine materials that are mixtures of multiple components. Various methods of chemical purification, e.g., chromatography, are used to isolate analytes from mixtures prior to mass spectrometric analysis. However, these techniques take time and specialized instrumentation, both of which could potentially be obviated via the use of ultra-high-resolution mass spectrometry. Here we report on the use of Orbitrap™-based Fourier-transform mass spectrometry to perform isotope ratio measurements of single species within mixtures delivered to the mass spectrometer (MS) without prior chromatographic separation. We demonstrate that instrument biases (attributed here to space charge effects) within the Orbitrap mass analyzer can cause the measured ¹³C/¹²C ratio of a molecular ion in the presence of non-analyte-derived ‘contaminating’ species to spuriously decrease relative to the ¹³C/¹²C ratio measured for the same ion in a pure analyte. We observe that the decrease in ¹³C/¹²C is proportional to the relative concentrations of the additional ‘contaminating’ components. We then recommend several strategies by which this effect can be mediated such that accurate isotope ratios can be obtained

Toward Full Peptide Sequence Coverage by Dual Fragmentation Combining Electron-Transfer and Higher-Energy Collision Dissociation Tandem Mass Spectrometry

Increasing peptide sequence coverage by tandem mass spectrometry improves confidence in database search-based peptide identification and facilitates mapping of post-translational modifications and de novo sequencing. Inducing 2-fold fragmentation by combining electron-transfer and higher-energy collision dissociation (EThcD) generates dual fragment ion series and facilitates extensive peptide backbone fragmentation. After an initial electron-transfer dissociation step, all ions including the unreacted precursor ions are subjected to collision induced dissociation which yields b/y- and c/z-type fragment ions in a single spectrum. This new fragmentation scheme provides richer spectra and substantially increases the peptide sequence coverage and confidence in peptide identification

Complete Protein Characterization Using Top-Down Mass Spectrometry and Ultraviolet Photodissociation

The top-down approach to proteomics offers compelling advantages due to the potential to provide complete characterization of protein sequence and post-translational modifications. Here we describe the implementation of 193 nm ultraviolet photodissociation (UVPD) in an Orbitrap mass spectrometer for characterization of intact proteins. Near-complete fragmentation of proteins up to 29 kDa is achieved with UVPD including the unambiguous localization of a single residue mutation and several protein modifications on Pin1 (Q13526), a protein implicated in the development of Alzheimer’s disease and in cancer pathogenesis. The 5 ns, high-energy activation afforded by UVPD exhibits far less precursor ion-charge state dependence than conventional collision- and electron-based dissociation methods

Development of a GC/Quadrupole-Orbitrap Mass Spectrometer, Part I: Design and Characterization

Identification of unknown compounds is of critical importance in GC/MS applications (metabolomics, environmental toxin identification, sports doping, petroleomics, and biofuel analysis, among many others) and remains a technological challenge. Derivation of elemental composition is the first step to determining the identity of an unknown compound by MS, for which high accuracy mass and isotopomer distribution measurements are critical. Here, we report on the development of a dedicated, applications-grade GC/MS employing an Orbitrap mass analyzer, the GC/Quadrupole-Orbitrap. Built from the basis of the benchtop Orbitrap LC/MS, the GC/Quadrupole-Orbitrap maintains the performance characteristics of the Orbitrap, enables quadrupole-based isolation for sensitive analyte detection, and includes numerous analysis modalities to facilitate structural elucidation. We detail the design and construction of the instrument, discuss its key figures-of-merit, and demonstrate its performance for the characterization of unknown compounds and environmental toxins