Dataset Information

Performance evaluation of a dual linear ion trap-Fourier transform ion cyclotron resonance mass spectrometer for proteomics research.

ABSTRACT: A novel dual cell linear ion trap Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR MS) and its performance characteristics are reported. A linear ion trap-Fourier transform ion cyclotron resonance mass spectrometer has been modified to incorporate a LTQ-Velos mass spectrometer. This modified instrument features efficient ion accumulation and fast MS/MS acquisition capabilities of dual cell linear RF ion trap instruments coupled to the high mass accuracy, resolution, and dynamic range of a FT-ICR for improved proteomic coverage. The ion accumulation efficiency is demonstrated to be an order of magnitude greater than that observed with LTQ-FT Ultra instrumentation. The proteome coverage with yeast was shown to increase over the previous instrument generation by 50% (100% increase on the peptide level). In addition, many lower abundance level yeast proteins were only detected with this modified instrument. This novel configuration also enables beam type CID fragmentation using a dual cell RF ion trap mass spectrometer. This technique involves accelerating ions between traps while applying an elevated DC offset to one of the traps to accelerate ions and induce fragmentation. This instrument design may serve as a useful option for labs currently considering purchasing new instrumentation or upgrading existing instruments.

Biological significance

A novel hybrid mass spectrometer that allows increased MS/MS acquisition rates with high mass measurement accuracy and new ion fragmentation methods greatly improves the number of proteins, posttranslational modifications and protein-protein interactions that can be identified from cells.

SUBMITTER: Weisbrod CR

PROVIDER: S-EPMC3972134 | biostudies-literature | 2013 Aug

REPOSITORIES: biostudies-literature

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Performance evaluation of a dual linear ion trap-Fourier transform ion cyclotron resonance mass spectrometer for proteomics research.

Weisbrod Chad R CR Hoopmann Michael R MR Senko Michael W MW Bruce James E JE

Journal of proteomics 20130413

A novel dual cell linear ion trap Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR MS) and its performance characteristics are reported. A linear ion trap-Fourier transform ion cyclotron resonance mass spectrometer has been modified to incorporate a LTQ-Velos mass spectrometer. This modified instrument features efficient ion accumulation and fast MS/MS acquisition capabilities of dual cell linear RF ion trap instruments coupled to the high mass accuracy, resolution, and dynami ...[more]

PMID: 23590889

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Project description:Metabolomics is an interdisciplinary field that aims to study all metabolites < 1500 Da that are ubiquitously found within all organisms. Metabolomics is experiencing exponential growth and commonly relies on high-resolution mass spectrometry (HRMS). Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) is a form of HRMS that is particularly well suited for metabolomics research due to its exceptionally high resolution (105-106) and sensitivity with a mass accuracy in parts per billion (ppb). In this regard, FT-ICR-MS can provide valuable insights into the metabolomics analysis of complex biological systems due to unique capabilities such as the easy separation of isobaric and isomeric species, isotopic fine structure analysis, spatial resolution of metabolites in cells and tissues, and a high confidence (<1 ppm mass error) in metabolite identification. Alternatively, the large and complex data sets, long acquisition times, high cost, and limited access mainly through national mass spectrometry facilities may impede the routine adoption of FT-ICR-MS by metabolomics researchers. This review examines recent applications of FT-ICR-MS metabolomics in the search for clinical and non-human biomarkers; for the analysis of food, beverage, and environmental samples; and for the high-resolution imaging of tissues and other biological samples. We provide recent examples of metabolomics studies that highlight the advantages of FT-ICR-MS for the detailed and reliable characterization of the metabolome. Additionally, we offer some practical considerations for implementing FT-ICR-MS into a research program by providing a list of FT-ICR-MS facilities and by identifying different high-throughput interfaces, varieties of sample types, analysis methods (e.g., van Krevelen diagrams, Kendrick mass defect plot, etc.), and sample preparation and handling protocols used in FT-ICR-MS experiments. Overall, FT-ICR-MS holds great promise as a vital research tool for advancing metabolomics investigations.

Project description:The reactivity of 10 charged phenyl radicals toward several amino acids was examined in the gas phase in a dual-cell Fourier transform ion cyclotron resonance mass spectrometer. All radicals abstract a hydrogen atom from the amino acids, as expected. The most electrophilic radicals (with the greatest calculated vertical electron affinities (EA) at the radical site) also react with these amino acids via NH(2) abstraction (a nonradical nucleophilic addition-elimination reaction). Both the radical (hydrogen atom abstraction) and nonradical (NH(2) abstraction) reaction efficiencies were found to increase with the electrophilicity (EA) of the radical. However, NH(2) abstraction is more strongly influenced by EA. In contrast to an earlier report, the ionization energies of the amino acids do not appear to play a general reactivity-controlling role. Studies using several partially deuterium-labeled amino acids revealed that abstraction of a hydrogen atom from the α-carbon is only preferred for glycine; for the other amino acids, a hydrogen atom is preferentially abstracted from the side chain. The electrophilicity of the radicals does not appear to have a major influence on the site from which the hydrogen atom is abstracted. Hence, the regioselectivity of hydrogen atom abstraction appears to be independent of the structure of the radical but dependent on the structure of the amino acid. Surprisingly, abstraction of two hydrogen atoms was observed for the N-(3-nitro-5-dehydrophenyl)pyridinium radical, indicating that substituents on the radical not only influence the EA of the radical but also can be involved in the reaction. In disagreement with an earlier report, proline was found to display several unprecedented reaction pathways that likely do not proceed via a radical mechanism but rather by a nucleophilic addition-elimination mechanism. Both NH(2) and (15)NH(2) groups were abstracted from lysine labeled with (15)N on the side chain, indicating that NH(2) abstraction occurs both from the amino terminus and from the side chain. Quantum chemical calculations were employed to obtain insights into some of the reaction mechanisms.

Project description:Mass spectrometry (MS) based top-down proteomics provides rich information about proteoforms arising from combinatorial amino acid sequence variations and post-translational modifications (PTMs). Fourier transform ion cyclotron resonance (FT-ICR) MS affords ultrahigh resolving power and provides high-accuracy mass measurements, presenting a powerful tool for top-down MS characterization of proteoforms. However, the detection and characterization of large proteins from complex mixtures remain challenging due to the exponential decrease in S: N with increasing molecular weight (MW) and coeluting low-MW proteins; thus, size-based fractionation of complex protein mixtures prior to MS analysis is necessary. Here, we directly combine MS-compatible serial size exclusion chromatography (sSEC) fractionation with 12 T FT-ICR MS for targeted top-down characterization of proteins from complex mixtures extracted from human and swine heart tissue. Benefiting from the ultrahigh resolving power of FT-ICR, we isotopically resolved 31 distinct proteoforms (30-50 kDa) simultaneously in a single mass spectrum within a 100 m/ z window. Notably, within a 5 m/ z window, we obtained baseline isotopic resolution for 6 distinct large proteoforms (30-50 kDa). The ultrahigh resolving power of FT-ICR MS combined with sSEC fractionation enabled targeted top-down analysis of large proteoforms (>30 kDa) from the human heart proteome without extensive chromatographic separation or protein purification. Further separation of proteoforms inside the mass spectrometer (in-MS) allowed for isolation of individual proteoforms and targeted electron capture dissociation (ECD), yielding high sequence coverage. sSEC/FT-ICR ECD facilitated the identification and sequence characterization of important metabolic enzymes. This platform, which facilitates deep interrogation of proteoform primary structure, is highly tunable, allows for adjustment of MS and MS/MS parameters in real time, and can be utilized for a variety of complex protein mixtures.

Dataset Information

Performance evaluation of a dual linear ion trap-Fourier transform ion cyclotron resonance mass spectrometer for proteomics research.

Biological significance

Publications

Performance evaluation of a dual linear ion trap-Fourier transform ion cyclotron resonance mass spectrometer for proteomics research.

Similar Datasets

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