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This work describes a novel mass spectrometer coupled to gas chromatography (GC-MS) that simultaneously displays the mass spectral information of electron (EI)- and chemical ionization (CI)-generated ion populations for a single chromatographic peak. After GC separation, the eluent is equally split and supplied in parallel to an EI and a novel CI source, both operating continuously. Precise switching of the ion optics provides the exact timing to consecutively extract the respective ion population from both sources and transfer them into a time-of-flight (TOF) mass analyzer. This technique enables the acquisition of complementary information from both ion populations (EI and CI) within a single chromatographic run and with sufficient data points to retain the chromatographic fidelity. The carefully designed GC transfer setup, fast ion optical switching, and synchronized TOF data acquisition system provide an automatic and straightforward spectral alignment of two ion populations. With an eluent split ratio of about 50% between the two ion sources, instrument detection limits of <40 fg on the column (octafluoronaphthalene) for the EI and <2 pg (benzophenone) for the CI source were obtained. The system performance and the additional analytical value for compound identification are demonstrated by means of different common GC standard mixtures and a commercial perfume sample of unknown composition.
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Electrones , Cromatografía de Gases y Espectrometría de Masas/métodos , Espectrometría de MasasRESUMEN
We evaluate the performance of a new chemical ionization source called Vocus, consisting of a discharge reagent-ion source and focusing ion-molecule reactor (FIMR) for use in proton-transfer-reaction time-of-flight mass spectrometry (PTR-TOF) measurements of volatile organic compounds (VOCs) in air. The reagent ion source uses a low-pressure discharge. The FIMR consists of a glass tube with a resistive coating, mounted inside a radio frequency (RF) quadrupole. The axial electric field is used to enhance ion collision energies and limit cluster ion formation. The RF field focuses ions to the central axis of the reactor and improves the detection efficiency of product ions. Ion trajectory calculations demonstrate the mass-dependent focusing of ions and enhancement of the ion collision energy by the RF field, in particular for the lighter ions. Product ion signals are increased by a factor of 10 when the RF field is applied (5000-18â¯000 cps ppbv-1), improving measurement precision and detection limits while operating at very similar reaction conditions as traditional PTR instruments. Because of the high water mixing ratio in the FIMR, we observe no dependence of the sensitivity on ambient sample humidity. In this work, the Vocus is interfaced to a TOF mass analyzer with a mass resolving power up to 12â¯000, which allows clear separation of isobaric ions, observed at nearly every nominal mass when measuring ambient air. Measurement response times are determined for a range of ketones with saturation vapor concentrations down to 5 × 104 µg m-3 and compare favorably with previously published results for a PTR-MS instrument.
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Hemoglobin disorder diagnosis is a complex procedure combining several analytical steps. Due to the lack of specificity of the currently used protein analysis methods, the identification of uncommon hemoglobin variants (proteoforms) can become a hard task to accomplish. The aim of this work was to develop a mass spectrometry-based approach to quickly identify mutated protein sequences within globin chain variants. To reach this goal, a top-down electron transfer dissociation mass spectrometry method was developed for hemoglobin ß chain analysis. A diagnostic product ion list was established with a color code strategy allowing to quickly and specifically localize a mutation in the hemoglobin ß chain sequence. The method was applied to the analysis of rare hemoglobin ß chain variants and an (A)γ-ß fusion protein. The results showed that the developed data analysis process allows fast and reliable interpretation of top-down electron transfer dissociation mass spectra by nonexpert users in the clinical area.
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Hemoglobinas/análisis , Espectrometría de Masas/métodos , Secuencia de Aminoácidos , Hemoglobina Fetal/análisis , Hemoglobina Fetal/genética , Fusión Génica , Variación Genética , Hemoglobinas/genética , Hemoglobinas Anormales/análisis , Hemoglobinas Anormales/genética , Humanos , Datos de Secuencia Molecular , Mutación , Espectrometría de Masas en Tándem/métodos , Flujo de Trabajo , Globinas beta/análisis , Globinas beta/genéticaRESUMEN
This work presents a newly designed energy-resolving time-of-flight mass spectrometer (E-TOFMS) for analysing the energy and mass of ions in bulk plasma. The system comprises an electrostatic sector analyser (ESA) for energy-to-charge (E/Q) ratio resolution and an orthogonal reflectron TOFMS for mass-to-charge (m/Q) ratio analysis. The design choices are explained, providing insight into electron and ion path simulations. The instrument was characterised using various ion generation sources, including an electron impact ion source, high power impulse magnetron sputtering, and microwave plasma electron cyclotron resonance sources. To validate its functionality, the energy-resolving data was compared with data obtained under the same conditions using a Langmuir probe and a retarding field energy analyser (RFEA). The benefits of the proposed E-TOFMS were demonstrated by sputtering highly alloyed steel with multiple isotope-rich elements, such as Mo or W. This technique offers an E/Q ratio resolution of up to 0.15 V for a range up to 125 V and a m/Q ratio resolution of at least 700 Th for a range up to 250 Th, with a temporal resolution of 10 µs.
RESUMEN
Electron transfer dissociation (ETD)-based top-down mass spectrometry (MS) is the method of choice for in-depth structure characterization of large peptides, small- and medium-sized proteins, and non-covalent protein complexes. Here, we describe the performance of this approach for structural analysis of intact proteins as large as the 80 kDa serotransferrin. Current time-of-flight (TOF) MS technologies ensure adequate resolution and mass accuracy to simultaneously analyze intact 30-80 kDa protein ions and the complex mixture of their ETD product ions. Here, we show that ETD TOF MS is efficient and may provide extensive sequence information for unfolded and highly charged (around 1 charge/kDa) proteins of ~30 kDa and structural motifs embedded in larger proteins. Sequence regions protected by disulfide bonds within intact non-reduced proteins oftentimes remain uncharacterized due to the low efficiency of their fragmentation by ETD. For serotransferrin, reduction of S-S bonds leads to significantly varied ETD fragmentation pattern with higher sequence coverage of N- and C-terminal regions, providing a complementary structural information to top-down analysis of its oxidized form.
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Anhidrasas Carbónicas/química , Péptidos/química , Espectrometría de Masas en Tándem/instrumentación , Transferrinas/química , Secuencia de Aminoácidos , Animales , Bovinos , Transporte de Electrón , Electrones , Diseño de Equipo , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Oxidación-ReducciónRESUMEN
Improving qualitative and quantitative characterization of monoclonal antibodies is essential, because of their increasing popularity as therapeutic drug targets. Electron transfer dissociation (ETD)-based top-down mass spectrometry (MS) is the method of choice for in-depth characterization of post-translationally modified large peptides, small- and medium-sized proteins, and noncovalent protein complexes. Here, we describe the performance of ETD-based top-down mass spectrometry for structural analysis of intact 150 kDa monoclonal antibodies, immunoglobulins G (IgGs). Simultaneous mass analysis of intact IgGs as well as a complex mixture of ETD product ions at sufficiently high resolution and mass accuracy in a wide m/z range became possible because of recent advances in state-of-the-art time-of-flight (TOF) mass spectrometry. High-resolution ETD TOF MS performed on IgG1-kappa from murine myeloma cells and human anti-Rhesus D IgG1 resulted in extensive sequence coverage of both light and heavy chains of IgGs and revealed information on their variable domains. Results are superior and complementary to those previously generated by collision-induced dissociation. However, numerous disulfide bonds drastically reduce the efficiency of top-down ETD fragmentation within the protected sequence regions, leaving glycosylation uncharacterized. Further increases in the experiment sensitivity and improvement of ion activation before and after ETD reaction are needed to target S-S bond-protected sequence regions and post-translational modifications.
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Anticuerpos Monoclonales/química , Espectrometría de Masa por Ionización de Electrospray , Secuencia de Aminoácidos , Animales , Línea Celular , Transporte de Electrón , Humanos , Inmunoglobulina G/química , Ratones , Datos de Secuencia Molecular , Procesamiento Proteico-PostraduccionalRESUMEN
Liquid chromatography (LC) electron transfer dissociation (ETD) tandem mass spectrometry (MS/MS) of protein digests is demonstrated in a hybrid quadrupole-hexapole orthogonal time-of-flight (OTOF) mass spectrometer. Analyte ions are selected in a mass-analyzing quadrupole, accumulated in the hexapole linear ETD reaction cell and mutually stored with ETD reagent anions. Product ions are collected in an ion cooler and then analyzed by an OTOF mass analyzer. The hexapole structure of the ETD reaction cell allows for a broad fragment ion mass range distribution and a high ion storage capacity. Analytically useful ETD OTOF-MS/MS spectra could be obtained at a rate of faster than 2 Hz. When used in conjunction with LC this high speed allows for several MS and MS/MS spectra to be obtained across each LC peak. An MS scan is used to select the precursor ions. With a 1 m flight tube and single reflection, resolutions of about 10 k and a mass accuracy of 5 ppm were achieved. When analyzing a 100 fmol solution of a tryptic digest of bovine serum albumin (BSA) by LC/ETD MS/MS, 27 unique peptides were identified with a summed Mascot score of 1316 using the Swiss Prot database. In addition, we explored the capability for analyzing small proteins with the present hybrid instrument. ETD MS/MS of intact ubiquitin ([M+12H](12+)) leads to the identification of the protein with a Mascot score of 264.
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Cromatografía Liquida/métodos , Proteínas/análisis , Proteínas/metabolismo , Espectrometría de Masas en Tándem/instrumentación , Espectrometría de Masas en Tándem/métodos , Secuencia de Aminoácidos , Animales , Bovinos , Cromatografía Liquida/economía , Electrones , Diseño de Equipo , Datos de Secuencia Molecular , Péptidos/análisis , Péptidos/metabolismo , Albúmina Sérica Bovina/análisis , Albúmina Sérica Bovina/metabolismo , Espectrometría de Masas en Tándem/economía , Factores de Tiempo , Tripsina/metabolismo , Ubiquitina/análisis , Ubiquitina/metabolismoRESUMEN
Electron transfer dissociation (ETD) of proteins is demonstrated in a hybrid quadrupole-hexapole Fourier transform ion cyclotron resonance mass spectrometer (Qh-FTICRMS). Analyte ions are selected in the mass analyzing quadrupole, accumulated in the hexapole linear ion trap, reacted with fluoranthene reagent anions, and then analyzed via an FTICR mass analyzer. The hexapole trap allows for a broad fragment ion mass range and a high ion storage capacity. Using a 3 T FTICRMS, resolutions of 60 000 were achieved with mass accuracies averaging below 1.4 ppm. The high resolution, high mass accuracy ETD spectra provided by FTICR obviates the need for proton transfer reaction (PTR) charge state reduction of ETD product ions when analyzing proteins or large peptides. This is demonstrated with the ETD of ubiquitin and apomyoglobin yielding sequence coverages of 37 and 20%, respectively. We believe this represents the first reported successful combination of ETD and a FTICRMS.