Pimp My Spec!

The National High Magnetic Field Laboratory (NHMFL) has identified as one of its next high-field persistent-magnet targets a 21 T superconducting magnet system to be used in a Fourier Transform Ion Cyclotron Resonance (FT-ICR) Mass Spectrometer. The required fundamental parameters of a 21 T central field and 110 mm warm bore diameter are similar to those already successfully demonstrated in the NHMFL's Ultra-Wide Bore 900 MHz Nuclear Magnetic Resonance magnet system. However, there are several ancillary requirements of the FT-ICR spectrometer that require technical attention such as fringe field restrictions requiring shielding, length of uniformity zone, magnitude of inhomogeneity, horizontal orientation of the magnet axis and a limitation on the length of the cryostat. This paper describes the overall system requirements, features and status of the superconducting magnet conceptual design proposed for meeting these requirements

Arginine-vasopressin (AVP) and lysine-vasopressin (LVP) were analyzed by reversed-phase liquid chromatography/mass spectrometry (LC-MS) using Fourier-transform ion cyclotron resonance (FT-ICR) mass spectrometry (MS) electrospray ionization (ESI) in the positive ion mode. LVP and AVP exhibited the protonated adduct [M+H](+) as the predominant ion at m/z 1056.43965 and at m/z 1084.44561, respectively. Infrared multiphoton dissociation (IRMPD), using a CO(2) laser source at a wavelength of 10.6 μm, was applied to protonated vasopressin molecules. The IRMPD mass spectra presented abundant mass fragments essential for a complete structural information. Several fragment ions, shared between two target molecules, are discussed in detail. Some previously unpublished fragments were identified unambiguously utilizing the high resolution and accurate mass information provided by the FT-ICR mass spectrometer. The opening of the disulfide loop and the cleavage of the peptide bonds within the ring were observed even under low-energy fragmentation conditions. Coupling the high-performance FT-ICR mass spectrometer with IRMPD as a contemporary fragmentation technique proved to be very promising for the structural characterization of vasopressin.

Mass spectrometry-based unbiased analysis of the full complement of secretory peptides is expected to facilitate the identification of unknown biologically active peptides. However, tandem MS sequencing of endogenous peptides in their native form has proven difficult because they show size heterogeneity and contain multiple internal basic residues, the characteristics not found in peptide fragments produced by in vitro digestion. Endogenous peptides remain largely unexplored by electron transfer dissociation (ETD), despite its widespread use in bottom-up proteomics. We used ETD, in comparison to collision induced dissociation (CID), to identify endogenous peptides derived from secretory granules of a human endocrine cell line. For mass accuracy, both MS and tandem MS were analyzed on an Orbitrap. CID and ETD, performed in different LC-MS runs, resulted in the identification of 795 and 569 unique peptides (ranging from 1000 to 15000 Da), respectively, with an overlap of 397. Peptides larger than 3000 Da accounted for 54% in CID and 46% in ETD identifications. Although numerically outperformed by CID, ETD provided more extensive fragmentation, leading to the identification of peptides that are not reached by CID. This advantage was demonstrated in identifying a new antimicrobial peptide from neurosecretory protein VGF (non-acronymic), VGF[554-577]-NH2, or in differentiating nearly isobaric peptides (mass difference less than 2 ppm) that arise from alternatively spliced exons of the gastrin-releasing peptide gene. CID and ETD complemented each other to add to our knowledge of the proteolytic processing sites of proteins implicated in the regulated secretory pathway. An advantage of the use of both fragmentation methods was also noted in localization of phosphorylation sites. These findings point to the utility of ETD mass spectrometry in the global study of endogenous peptides, or peptidomics.

Metabolic profiling by FT-ICR-MS and ESI-Q-TOF-MS is an effective method to capture a rough image of metabolism. It should be performed after the accuracy is confirmed by using the authentic sample after an elaborate calibration is done, when an individual metabolite is identified from the accurate mass for FT-ICR-MS. Moreover, when ESI-Q-TOF-MS is used, the CID spectrum should also be measured, and compared with that of the authentic sample.

Initial results obtained by incorporation of an inductively coupled plasma (ICP) ion source with a Fourier transform ion cyclotron resonance (FTICR) mass spectrometer are reported, along with spectra which demonstrate sub-milligram-per-liter detection limits and high mass resolution (mass resolving power, m/Δm10%V = 7000−10 000). Also shown is an ICP-FTICR spectrum with ultrahigh resolving power (m/Δm10%V = 88 000). Both instrumental configurations used to conduct this work, one at the University of Florida, and the other at the National High Magnetic Field Laboratory, are described. Also included are concise results from a computer aided interference study designed to enumerate mass spectral interferences requiring mass resolving power in excess of 10 000.

An electron injection system based on an indirectly heated ring-shaped dispenser cathode has been developed and installed in a 7 Tesla Fourier transform ion cyclotron resonance (FTICR) mass spectrometer. This new hardware design allows high-rate electron capture dissociation (ECD) to be carried out by a hollow electron beam coaxial with the ion cyclotron resonance (ICR) trap. Infrared multiphoton dissociation (IRMPD) can also be performed with an on-axis IR-laser beam passing through a hole at the centre of the dispenser cathode. Electron and photon irradiation times of the order of 100 ms are required for efficient ECD and IRMPD, respectively. As ECD and IRMPD generate fragments of different types (mostly c, z and b, y, respectively), complementary structural information that improves the characterization of peptides and proteins by FTICR mass spectrometry can be obtained. The developed technique enables the consecutive or simultaneous use of the ECD and IRMPD methods within a single FTICR experimental sequence and on the same ensemble of trapped ions in multistage tandem (MS/MS/MS or MS(n)) mass spectrometry. Flexible changing between ECD and IRMPD should present advantages for the analysis of protein digests separated by liquid chromatography prior to FTICRMS. Furthermore, ion activation by either electron or laser irradiation prior to, as well as after, dissociation by IRMPD or ECD increases the efficiency of ion fragmentation, including the w-type fragment ion formation, and improves sequencing of peptides with multiple disulfide bridges. The developed instrumental configuration is essential for combined ECD and IRMPD on FTICR mass spectrometers with limited access into the ICR trap.

The six high-affinity insulin-like growth factor-binding proteins (IGFBPs) comprise a conserved family of secreted molecules that modulate IGF actions by regulating their half-life and access to signaling receptors, and also exert biological effects that are independent of IGF binding. IGFBPs are composed of cysteine-rich amino- (N-) and carboxyl- (C-) terminal domains, along with a cysteine-poor central linker segment. IGFBP-5 is the most conserved IGFBP, and contains 18 cysteines, but only 2 of 9 putative disulfide bonds have been mapped to date. Using a mass spectrometry (MS)-based strategy combining sequential electron transfer dissociation (ETD) and collision-induced dissociation (CID) steps, in which ETD fragmentation preferentially induces cleavage of disulfide bonds, and CID provides exact disulfide linkage assignments between liberated peptides, we now have definitively mapped 5 disulfide bonds in IGFBP-5. In addition, in conjunction with ab initio molecular modeling we are able to assign the other 4 disulfide linkages to within a GCGCCXXC motif that is conserved in five IGFBPs. Because of the nature of ETD fragmentation MS experiments were performed without chemical reduction of IGFBP-5. Our results not only establish a disulfide bond map of IGFBP-5 but also define a general approach that takes advantage of the specificity of ETD and the scalability of tandem MS, and the predictive power of ab initio molecular modeling to characterize unknown disulfide linkages in proteins.

FT-ICR MS optimization for the analysis of intact proteins

Molecular properties of deep-sea dissolved organic matter are predictable by the central limit theorem: Evidence from tandem FT-ICR-MS

Fourier-transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) is one of the state-of-the-art methods to analyze complex natural organic mixtures. The precision of detected masses is crucial for molecular formula attribution. Random errors can be reduced by averaging multiple measurements of the same mass, but because of limited availability of ultrahigh-resolution mass spectrometers, most studies cannot afford analyzing each sample multiple times. Here we show that random errors can be eliminated also by averaging mass spectral data from independent environmental samples. By averaging the spectra of 30 samples analyzed on our 15 T instrument we reach a mass precision comparable to a single spectrum of a 21 T instrument. We also show that it is possible to accurately and reproducibly determine isotope ratios with FT-ICR-MS. Intensity ratios of isotopologues were improved to a degree that measured deviations were within the range of natural isotope fractionation effects. In analogy to δ13C in environmental studies, we propose Δ13C as an analytical measure for isotope ratio deviances instead of widely employed C deviances. In conclusion, here we present a simple tool, extensible to Orbitrap-based mass spectrometers, for postdetection data processing that significantly improves mass accuracy and the precision of intensity ratios of isotopologues at no extra cost.

<p>Plant-microbe interplay in the rhizosphere generates multi-faceted chemical gradients. The soil solution is a crucial component of the rhizosphere, where chemical gradients of organic molecules first develop upon growth of roots, introduction of plant-derived carbon and microbial turnover. Studying these gradients requires high resolution both in time and space as well as high chemical specificity to resolve the multitude of compounds. Existing methods to probe the rhizosphere soil solution were mostly limited to bulk chemical parameters, inorganic ions or targeted analysis of organic molecules. However, to decipher organic carbon turnover in the rhizosphere the characterization of the complex pool of soil solution organic matter is needed.</p><p>Here we present a novel method that combines time-resolved collection of soil solution samples via micro-suction cups in the rhizosphere with ultrahigh chemical resolution provided by Fourier-transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) to unravel developing pattern of soil solution organic matter.</p><p>Zea mays plants were grown in soil columns for three weeks and soil solution samples of undisturbed root-soil system were collected once a week. Growth of the root system and hence position of sampling locations in relation to the distance from the root, were followed by X-ray computed tomography (X-ray CT).</p><p>The online sample preparation was optimised to extract and desalt the organic matter from a few microliters of soil solution. The downscaling to a nano-liquid chromatography system for the on-line extraction allowed the analysis of only minute amounts of organic carbon within the samples (down to 10 ng). Given the high background concentration of soil-derived organic carbon, the high mass resolution and sensitivity of FT-ICR-MS enabled to distinguish root derived molecules from soil organic matter based on their exact masses. Molecular formulas of the root derived molecules could be calculated showing distinct chemical characteristics as compared to the bulk soil solution. X-ray CT analyses enabled relating the results from the chemical analysis to distance from the root and root age. With increasing influence of the roots higher molecular masses and an increasing degree of oxygenation of the molecules could be observed.</p><p>Our method is thus capable to show the changing small scale pattern of soil solution organic matter during the early rhizosphere development, closing the knowledge gap between root exudates, soil chemistry and microbial processes.</p>

Ultrasensitive nanoscale proteomics approaches for characterizing proteins from complex proteomic samples of <50 ng of total mass are described. Protein identifications from 0.5 pg of whole proteome extracts were enabled by ultrahigh sensitivity (<75 zmol for individual proteins) achieved using high-efficiency (peak capacities of approximately 10(3)) 15-microm-i.d. capillary liquid chromatography separations (i.e., using nanoLC, approximately 20 nL/min mobile-phase flow rate at the optimal linear velocity of approximately 0.2 cm/s) coupled on-line with a micro-solid-phase sample extraction and a nanoscale electrospray ionization interface to a 11.4-T Fourier transform ion cyclotron resonance (FTICR) mass spectrometer (MS). Proteome measurement coverage improved as sample size was increased from as little as 0.5 pg of sample. It was found that a 2.5-ng sample provided 14% coverage of all annotated open reading frames for the microorganism Deinococcus radiodurans, consistent with previous results for a specific culture condition. The estimated detection dynamic range for detected proteins was 10(5)-10(6). An improved accurate mass and LC elution time two-dimensional data analysis methodology, used to both speed and increase the confidence of peptide/protein identifications, enabled identification of 872 proteins/run from a single 3-h nanoLC/FTICR MS analysis. The low-zeptomole-level sensitivity provides a basis for extending proteomics studies to smaller cell populations and potentially to a single mammalian cell. Application with ion trap MS/MS instrumentation allowed protein identification from 50 pg (total mass) of proteomic samples (i.e., approximately 100 times larger than FTICR MS), corresponding to a sensitivity of approximately 7 amol for individual proteins. Compared with single-stage FTICR measurements, ion trap MS/MS provided a much lower proteome measurement coverage and dynamic range for a given analysis time and sample quantity.

Compared with earlier methods of food characterization, metabolomics has brought an unprecedented boost in uncovering the wide biochemical composition of various food products. This change has only been possible through the advent of, and continuous innovations in, modern metabolomics platforms, especially mass spectrometry (MS). In MS, the most widely used technique is liquid chromatography coupled to MSn [i.e. LC–MSn (where n=variants of MS platforms required for separation of the sample)] followed by gas chromatography coupled to MSn (GC–MSn) and others like Fourier transform-ion cyclotron resonance mass spectrometry (FT-ICR-MS). The most critical steps in food metabolomics are sample preparation, extraction of metabolites, running, and data analysis. Apart from the biochemical characterization of food products, the emphasis has also been given to food safety and authenticity, characterizing food products during fermentation and processing, identifying health-related dietary biomarkers, and heath-friendly bioactive components through intervention studies of population cohorts. Food metabolomics in the future will play an important role in defining diets in a more precise way and helping to link the effects of diet and human nutrition. Thus, in the future, it will gather more interest from nutrition biologists to classify human diets broadly.

The study of protein kinetics requires an accurate measurement of isotopic ratios of peptides. Although Fourier transform-ion cyclotron resonance (FT-ICR) mass spectrometers yield accurate mass measurements of analytes, the isotopologue ratios are consistently lower than predicted. Recently, we demonstrated that the magnitude of the spectral error in the FT-ICR mass spectrometer is proportional to the scan duration of ions. Here, we present a novel isotopic ratio extrapolation (IRE) method for obtaining accurate isotopic ratio measurements. Accuracy is achieved by performing scans with different duration and extrapolation of the data to the initial moment of the ion rotation; IRE minimizes the absolute isotopic ratio error to ≤1%. We demonstrate the application of IRE in protein turnover studies using (2)H(2)O-metabolic labeling. Overall, this technique allows accurate measurements of the isotopic ratios of proteolytic peptides, a critical step for enabling routine studies of proteome dynamics.

Beach wrack is an important supplier of nutrients and organic matter to sandy beach ecosystems and underlying subterranean estuaries (STEs), producing metabolic hotspots in these otherwise organic carbon- and nutrient-poor environments. To assess the impact of beach wrack type (e.g., marine, terrestrial, plant, animal) and environmental settings (e.g., tidal inundation, precipitation, and solar irradiation) on nutrient and dissolved organic matter (DOM) release, a series of leaching experiments was conducted. Quantities of leached nutrients and dissolved organic carbon (DOC) were determined, and DOM molecular composition was investigated using Fourier-transform ion cyclotron resonance mass spectrometry (FT-ICR-MS). Millimolar—to molar amounts of DOC and dissolved nitrogen were released from the beach cast per kg dry weight, with type of wrack and leaching medium (fresh- vs. saltwater) exerting the biggest influences. Exemplary for animal cast, jellyfish leached up to two 100-fold more, mostly organic, nitrogen compared to all other beach wrack types. FT-ICR-MS data of solid-phase extracted DOM indicated that beach wrack releases compounds with putative mono- and oligosaccharide-, amino acid- and vitamin-type molecular formulae, which likely serve as valuable substrate for heterotrophic microorganisms. DOM from the brown seaweed Fucus sp. was more aromatic than seawater DOM and even beach wrack of terrestrial origin, probably from structural components and secondary metabolites such as phlorotannins. We conclude that DOM and nutrient release from beach wrack strongly depends on wrack type and leaching medium, may obscure molecular provenance proxies (e.g., terrestrial indices), and adds a nutritional boost to infiltrating sea- and rainwater which likely impact microbial respiration rates in the STE.