Large-Scale Interlaboratory DI-FT-ICR MS Comparability Study Employing Various Systems.

Ultrahigh resolution mass spectrometry (UHR-MS) coupled with direct infusion (DI) electrospray ionization offers a fast solution for accurate untargeted profiling. Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometers have been shown to produce a wealth of insights into complex chemical systems because they enable unambiguous molecular formula assignment even if the vast majority of signals is of unknown identity. Interlaboratory comparisons are required to apply this type of instrumentation in quality control (for food industry or pharmaceuticals), large-scale environmental studies, or clinical diagnostics. Extended comparisons employing different FT-ICR MS instruments with qualitative direct infusion analysis are scarce since the majority of detected compounds cannot be quantified. The extent to which observations can be reproduced by different laboratories remains unknown. We set up a preliminary study which encompassed a set of 17 laboratories around the globe, diverse in instrumental characteristics and applications, to analyze the same sets of extracts from commercially available standard human blood plasma and Standard Reference Material (SRM) for blood plasma (SRM1950), which were delivered at different dilutions or spiked with different concentrations of pesticides. The aim of this study was to assess the extent to which the outputs of differently tuned FT-ICR mass spectrometers, with different technical specifications, are comparable for setting the frames of a future DI-FT-ICR MS ring trial. We concluded that a cluster of five laboratories, with diverse instrumental characteristics, showed comparable and representative performance across all experiments, setting a reference to be used in a future ring trial on blood plasma.

Surface-Induced Dissociation of Protein Complexes in a Hybrid Fourier Transform Ion Cyclotron Resonance Mass Spectrometer.

Mass spectrometry continues to develop as a valuable tool in the analysis of proteins and protein complexes. In protein complex mass spectrometry studies, surface-induced dissociation (SID) has been successfully applied in quadrupole time-of-flight (Q-TOF) instruments. SID provides structural information on noncovalent protein complexes that is complementary to other techniques. However, the mass resolution of Q-TOF instruments can limit the information that can be obtained for protein complexes by SID. Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) provides ultrahigh resolution and ultrahigh mass accuracy measurements. In this study, an SID device was designed and successfully installed in a hybrid FT-ICR instrument in place of the standard gas collision cell. The SID-FT-ICR platform has been tested with several protein complex systems (homooligomers, a heterooligomer, and a protein-ligand complex, ranging from 53 to 85 kDa), and the results are consistent with data previously acquired on Q-TOF platforms, matching predictions from known protein interface information. SID fragments with the same m/z but different charge states are well-resolved based on distinct spacing between adjacent isotope peaks, and the addition of metal cations and ligands can also be isotopically resolved with the ultrahigh mass resolution available in FT-ICR.

2-Aminoethanol Extraction as a Method for Purifying Sc3N@C80 and for Differentiating Classes of Endohedral Fullerenes on the Basis of Reactivity.

Extraction with 2-aminoethanol is an inexpensive method for removing empty cage fullerenes from the soluble extract from electric-arc-generated fullerene soot that contains endohedral metallofullerenes of the type Sc3N@C2n (n = 34, 39, 40). Our method of separation exploits the fact that C60, C70, and other larger, empty cage fullerenes are more susceptible to nucleophilic attack than endohedral fullerenes and that these adducts can be readily extracted into 2-aminoethanol. This methodology has also been employed to examine the reactivity of the mixture of soluble endohedral fullerenes that result from doping graphite rods used in the Krätschmer-Huffman electric-arc generator with the oxides of Y, Lu, Dy, Tb, and Gd. For example, with Y2O3, we were able to detect by mass spectrometry several new families of endohedral fullerenes, namely Y3C108 to Y3C126, Y3C107 to Y3C125, Y4C128 to Y4C146, that resisted reactivity with 2-aminoethanol more than the empty cage fullerenes and the mono- and dimetallo fullerenes. The discovery of the family Y3C107 to Y3C125 with odd numbers of carbon atoms is remarkable, since fullerene cages must involve even numbers of carbon atoms. The newly discovered families of endohedral fullerenes with the composition M4C2n (M = Y, Lu, Dy, Tb, and Gd) are unusually resistant to reaction with 2-aminoethanol. Additionally, the individual endohedrals, Y3C112 and M3C102 (M = Lu, Dy, Tb and Gd), were remarkably less reactive toward 2-aminoethanol.

Pharmacokinetics of bisphenol A in humans following a single oral administration.

BACKGROUND: Human exposures to bisphenol A (BPA) are widespread. The current study addresses uncertainties regarding human pharmacokinetics of BPA. OBJECTIVE: To reduce uncertainties about the metabolism and excretion of BPA in humans following oral administration. METHODS: We exposed six men and eight women to 100 µg/kg bw of deuterated BPA (d6-BPA) by oral administration and conducted blood and urine analysis over a three day period. The use of d6-BPA allowed administered d6-BPA to be distinguished from background native (unlabeled) BPA. We calculated the rate of oral absorption, serum elimination, half-life, area under the curve (AUC), urinary excretion, and metabolism to glucuronide and sulfate conjugates. RESULTS: Mean serum total (unconjugated and conjugated) d6-BPA Cmax of 1711 nM (390 ng/ml) was observed at Tmax of 1.1 ± 0.50h. Unconjugated d6-BPA appeared in serum within 5-20 min of dosing with a mean Cmax of 6.5 nM (1.5 ng/ml) observed at Tmax of 1.3 ± 0.52 h. Detectable blood levels of unconjugated or total d6-BPA were observed at 48 h in some subjects at concentrations near the LOD (0.001-0.002 ng/ml). The half-times for terminal elimination of total d6-BPA and unconjugated d6-BPA were 6.4 ± 2.0 h and 6.2 ± 2.6h, respectively. Recovery of total administered d6-BPA in urine was 84-109%. Most subjects (10 of 14) excreted >90% as metabolites within 24h. CONCLUSIONS: Using more sensitive methods, our study expands the findings of other human oral pharmacokinetic studies. Conjugation reactions are rapid and nearly complete with unconjugated BPA comprising less than 1% of the total d6-BPA in blood at all times. Elimination of conjugates into urine largely occurs within 24h.

Application of Sholl analysis to quantify changes in growth and development in rat mammary gland whole mounts.

Studies that utilize the rodent mammary gland (MG) as an endpoint for assessing the developmental toxicity of chemical exposures typically employ either basic dimensional measurements or developmental scoring of morphological characteristics as a means to quantify MG development. There are numerous means by which to report these developmental changes, leading to inconsistent translation across laboratories. The Sholl analysis is a method historically used for quantifying neuronal dendritic patterns. The present study describes the use of the Sholl analysis to quantify MG branching characteristics. Using this method, we were able to detect significant differences in branching density in MG of peripubertal female Sprague Dawley rats that had been exposed to vehicle or a potent estrogen. These data suggest the Sholl analysis can be an effective tool for quantitatively measuring an important characteristic of MG development and for examining associations between MG growth and density and adverse effects in the breast.

Application of desorption electrospray ionization mass spectrometry imaging in breast cancer margin analysis.

Distinguishing tumor from normal glandular breast tissue is an important step in breast-conserving surgery. Because this distinction can be challenging in the operative setting, up to 40% of patients require an additional operation when traditional approaches are used. Here, we present a proof-of-concept study to determine the feasibility of using desorption electrospray ionization mass spectrometry imaging (DESI-MSI) for identifying and differentiating tumor from normal breast tissue. We show that tumor margins can be identified using the spatial distributions and varying intensities of different lipids. Several fatty acids, including oleic acid, were more abundant in the cancerous tissue than in normal tissues. The cancer margins delineated by the molecular images from DESI-MSI were consistent with those margins obtained from histological staining. Our findings prove the feasibility of classifying cancerous and normal breast tissues using ambient ionization MSI. The results suggest that an MS-based method could be developed for the rapid intraoperative detection of residual cancer tissue during breast-conserving surgery.

The first pilot project of the consortium for top-down proteomics: a status report.

Pilot Project #1--the identification and characterization of human histone H4 proteoforms by top-down MS--is the first project launched by the Consortium for Top-Down Proteomics (CTDP) to refine and validate top-down MS. Within the initial results from seven participating laboratories, all reported the probability-based identification of human histone H4 (UniProt accession P62805) with expectation values ranging from 10(-13) to 10(-105). Regarding characterization, a total of 74 proteoforms were reported, with 21 done so unambiguously; one new PTM, K79ac, was identified. Inter-laboratory comparison reveals aspects of the results that are consistent, such as the localization of individual PTMs and binary combinations, while other aspects are more variable, such as the accurate characterization of low-abundance proteoforms harboring >2 PTMs. An open-access tool and discussion of proteoform scoring are included, along with a description of general challenges that lie ahead including improved proteoform separations prior to mass spectrometric analysis, better instrumentation performance, and software development.

Resolving isotopic fine structure to detect and quantify natural abundance- and hydrogen/deuterium exchange-derived isotopomers.

Hydrogen/deuterium exchange (HDX) mass spectrometry (MS) is used for analyzing protein dynamics, protein folding/unfolding, and molecular interactions. Until this study, HDX MS experiments employed mass spectral resolving powers that afforded only one peak per nominal mass in a given peptide's isotope distribution, and HDX MS data analysis methods were developed accordingly. A level of complexity that is inherent to HDX MS remained unaddressed, namely, various combinations of natural abundance heavy isotopes and exchanged deuterium shared the same nominal mass and overlapped at previous resolving powers. For example, an A + 2 peak is comprised of (among other isotopomers) a two-(2)H-exchanged/zero-(13)C isotopomer, a one-(2)H-exchanged/one-(13)C isotopomer, and a zero-(2)H-exchanged/two-(13)C isotopomer. Notably, such isotopomers differ slightly in mass as a result of the ∼3 mDa mass defect between (2)H and (13)C atoms. Previous HDX MS methods did not resolve these isotopomers, requiring a natural-abundance-only (before HDX or "time zero") spectrum and data processing to remove its contribution. It is demonstrated here that high-resolution mass spectrometry can be used to detect isotopic fine structure, such as in the A + 2 profile example above, deconvolving the isotopomer species resulting from deuterium incorporation. Resolving isotopic fine structure during HDX MS therefore permits direct monitoring of HDX, which can be calculated as the sum of the fractional peak magnitudes of the deuterium-exchanged isotopomers. This obviates both the need for a time zero spectrum as well as data processing to account for natural abundance heavy isotopes, saving instrument and analysis time.

A rapid MALDI-TOF mass spectrometry workflow for Drosophila melanogaster differential neuropeptidomics.

BACKGROUND: Neuropeptides are a diverse category of signaling molecules in the nervous system regulating a variety of processes including food intake, social behavior, circadian rhythms, learning, and memory. Both the identification and functional characterization of specific neuropeptides are ongoing fields of research. Matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis of nervous tissues from a variety of organisms allows direct detection and identification of neuropeptides. Here, we demonstrate an analysis workflow that allows for the detection of differences in specific neuropeptides amongst a variety of neuropeptides being simultaneously measured. For sample preparation, we describe a straight-forward and rapid (minutes) method where individual adult Drosophila melanogaster brains are analyzed. Using a MATLAB-based data analysis workflow, also compatible with MALDI-TOF mass spectra obtained from other sample preparations and instrumentation, we demonstrate how changes in neuropeptides levels can be detected with this method. RESULTS: Over fifty isotopically resolved ion signals in the peptide mass range are reproducibly observed across experiments. MALDI-TOF MS profile spectra were used to statistically identify distinct relative differences in organ-wide endogenous levels of detected neuropeptides between biological conditions. In particular, three distinct levels of a particular neuropeptide, pigment dispersing factor, were detected by comparing groups of preprocessed spectra obtained from individual brains across three different D. melanogaster strains, each of which express different amounts of this neuropeptide. Using the same sample preparation, MALDI-TOF/TOF tandem mass spectrometry confirmed that at least 14 ion signals observed across experiments are indeed neuropeptides. Among the identified neuropeptides were three products of the neuropeptide-like precursor 1 gene previously not identified in the literature. CONCLUSIONS: Using MALDI-TOF MS and preprocessing/statistical analysis, changes in relative levels of a particular neuropeptide in D. melanogaster tissue can be statistically detected amongst a variety of neuropeptides. While the data analysis methods should be compatible with other sample preparations, the presented sample preparation method was sufficient to identify previously unconfirmed D. melanogaster neuropeptides.

Molecular imaging of drug transit through the blood-brain barrier with MALDI mass spectrometry imaging.

Drug transit through the blood-brain barrier (BBB) is essential for therapeutic responses in malignant glioma. Conventional methods for assessment of BBB penetrance require synthesis of isotopically labeled drug derivatives. Here, we report a new methodology using matrix assisted laser desorption ionization mass spectrometry imaging (MALDI MSI) to visualize drug penetration in brain tissue without molecular labeling. In studies summarized here, we first validate heme as a simple and robust MALDI MSI marker for the lumen of blood vessels in the brain. We go on to provide three examples of how MALDI MSI can provide chemical and biological insights into BBB penetrance and metabolism of small molecule signal transduction inhibitors in the brain - insights that would be difficult or impossible to extract by use of radiolabeled compounds.

Sc2(mu2-O) trapped in a fullerene cage: the isolation and structural characterization of Sc2(mu2-O)@C(s)6-C82 and the relevance of the thermal and entropic effects in fullerene isomer selection.

The new endohedral fullerene, Sc(2)(mu(2)-O)@C(s)(6)-C(82), has been isolated from the carbon soot obtained by electric arc generation of fullerenes utilizing graphite rods doped with 90% Sc(2)O(3) and 10% Cu (w/w). Sc(2)(mu(2)-O)@C(s)(6)-C(82) has been characterized by single crystal X-ray diffraction, mass spectrometry, and UV/vis spectroscopy. Computational studies have shown that, among the nine isomers that follow the isolated pentagon rule (IPR) for C(82), cage 6 with C(s) symmetry is the most favorable to encapsulate the cluster at T > 1200 K. Sc(2)(mu(2)-O)@C(s)(6)-C(82) is the first example in which the relevance of the thermal and entropic contributions to the stability of the fullerene isomer has been clearly confirmed through the characterization of the X-ray crystal structure.

Transformative effects of higher magnetic field in Fourier transform ion cyclotron resonance mass spectrometry.

The relationship of magnetic field strength and Fourier transform ion cyclotron resonance mass spectrometry performance was tested using three instruments with the same design but different fields of 4.7, 7, and 9.4 tesla. We found that the theoretically predicted "transformative" effects of magnetic field are indeed observed experimentally. The most striking effects were that mass accuracy demonstrated approximately second to third order improvement with the magnetic field, depending upon the charge state of the analyte, and that peak splitting, which prohibited automated data analysis at 4.7 T, was not observed at 9.4 T.

Negative electron transfer dissociation of glycosaminoglycans.

Structural characterization of glycosaminoglycans (GAGs) has been a challenge in the field of mass spectrometry, and the application of electron detachment dissociation (EDD) Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) has shown great promise to GAG oligosaccharide characterization in a single tandem mass spectrometry experiment. In this work, we apply the technique of negative electron transfer dissociation (NETD) to GAGs on a commercial ion trap mass spectrometer. NETD of GAGs, using fluoranthene or xenon as the reagent gas, produces fragmentation very similar to previously observed EDD fragmentation. Using fluoranthene or xenon, both glycosidic and cross-ring cleavages are observed, as well as even- and odd-electron products. The loss of SO(3) can be minimized and an increase in cross-ring cleavages is observed if a negatively charged carboxylate is present during NETD, which can be controlled by the charge state or the addition of sodium. NETD effectively dissociates GAGs up to eight saccharides in length, but the low resolution of the ion trap makes assigning product ions difficult. Similar to EDD, NETD is also able to distinguish the epimers iduronic acid from glucuronic acid in heparan sulfate tetrasaccharides and suggests that a radical intermediate plays an important role in distinguishing these epimers. These results demonstrate that NETD is effective at characterizing GAG oligosaccharides in a single tandem mass spectrometry experiment on a widely available mass spectrometry platform.

Negative electron transfer dissociation of deprotonated phosphopeptide anions: choice of radical cation reagent and competition between electron and proton transfer.

Despite significant developments in mass spectrometry technology in recent years, no routine proteomics sequencing tool is currently available for peptide anions. The use of a molecular open-shell cation is presented here as a possible reaction partner to induce electron transfer dissociation with deprotonated peptide anions. In this negative electron transfer dissociation (NETD) scheme, an electron is abstracted from the peptide anion and transferred to the radical cation. This is demonstrated for the example of the fluoranthene cation, C(16)H(10)(+*), which is reacted with deprotonated phosphorylated peptides in a 3-D ion trap mass spectrometer. Selective backbone cleavage at the C(alpha)-C bond is observed to yield a and x fragments, similarly to electron detachment dissociation (EDD) of peptide anions. Crucially, the phosphorylation site is left intact in the dissociation process, allowing an identification and localization of the post-translational modification (PTM) site. In contrast, NETD using Xe(+*) as the reagent cation results in sequential neutral losses (CO(2) and H(3)PO(4)) from a/x fragments, which complicate the interpretation of the mass spectra. This difference in dissociation behavior can be understood in the framework of the reduced recombination energy of the electron transfer process for fluoranthene, which is estimated at 2.5-4.5 eV, compared to 6.7-8.7 eV for xenon. Similarly to ETD, proton transfer is found to compete with electron transfer processes in NETD. Isotope fitting of the charge-reduced species shows that in the case of fluoranthene-mediated NETD, proton transfer only accounts for <20%, whereas this process highly abundant for Xe(+*) (43 and 82%). Since proton abstraction from Xe(+*) is not possible, this suggests that Xe(+*) ionizes other transient species in the ion trap, which then engage in proton transfer reactions with the peptide anions.

Structural characterization of intact proteins is enhanced by prevalent fragmentation pathways rarely observed for peptides.

While collisionally activated dissociation (CAD) pathways for peptides are well characterized, those of intact proteins are not. We systematically assigned CAD product ions of ubiquitin, myoglobin, and bovine serum albumin generated using high-yield, in-source fragmentation. Assignment of >98% of hundreds of product ions implies that the fragmentation pathways described are representative of the major pathways. Protein dissociation mechanisms were found to be modulated by both source declustering potential and precursor ion charge state. Like peptides, higher charge states of proteins fragmented at lower energies next to Pro, via mobile protons, while lower charge states fragmented at higher energies after Asp and Glu, via localized protons. Unlike peptides, however, predominant fragmentation channels of proteins occurred at intermediate charge states via non-canonical mechanisms and produced extensive internal fragmentation. The non-canonical mechanisms include prominent cleavages C-terminal to Pro and Asn, and N-terminal to Ile, Leu, and Ser; these cleavages, along with internal fragments, led to a 45% increase in sequence coverage, improving the specificity of top-down protein identification. Three applications take advantage of the different mechanisms of protein fragmentation. First, modulation of declustering potential selectively fragments different charge states, allowing the source region to be used as the first stage of a low-resolution tandem mass spectrometer, facilitating pseudo-MS(3) of product ions with known parent charge states. Second, development and integration of automated modulation of ion funnel declustering potential allows users access to a particular fragmentation mechanism, yielding facile cleavage on a liquid chromatography timescale. Third, augmentation of a top-down search engine improved protein characterization.

Physiologically based pharmacokinetic modeling of dibromoacetic acid in F344 rats.

A novel physiologically based pharmacokinetic (PBPK) model structure, which includes submodels for the common metabolites (glyoxylate (GXA) and oxalate (OXA)) that may be involved in the toxicity or carcinogenicity of dibromoacetic acid (DBA), has been developed. Particular attention is paid to the representation of hepatic metabolism, which is the primary elimination mechanism. DBA-induced suicide inhibition is modeled by irreversible covalent binding of the intermediate metabolite alpha-halocarboxymethylglutathione (alphaH1) to the glutathione-S-transferase zeta (GSTzeta) enzyme. We also present data illustrating the presence of a secondary non-GSTzeta metabolic pathway for DBA, but not dichloroacetic acid (DCA), that produces GXA. The model is calibrated with plasma and urine concentration data from DBA exposures in female F344 rats through intravenous (IV), oral gavage, and drinking water routes. Sensitivity analysis is performed to confirm identifiability of estimated parameters. Finally, model validation is performed with data sets not used during calibration. Given the structural similarity of dihaloacetates (DHAs), we hypothesize that the PBPK model presented here has the capacity to describe the kinetics of any member or mixture of members of this class in any species with the alteration of chemical-and species-specific parameters.

A seven atom cluster in a carbon cage, the crystallographically determined structure of Sc4(mu3-O)3@Ih-C80.

The tetrahedral array of four scandium atoms with oxygen atoms capping three of the four faces found in Sc(4)(mu(3)-O)(3)@I(h)-C(80) is the largest cluster isolated to date inside a fullerene cage.

Protein conformations can be probed in top-down HDX MS experiments utilizing electron transfer dissociation of protein ions without hydrogen scrambling.

Electron-transfer dissociation (ETD) is evaluated as a technique to provide local information on higher order structure and dynamics of a whole protein molecule. Isotopic labeling of highly flexible segments of a model 18 kDa protein is carried out in solution under mildly denaturing conditions by means of hydrogen/deuterium exchange (HDX), followed by transfer of intact protein ions to the gas phase by means of electrospray ionization, and mass-selection of a precursor ion for subsequent reactions with fluoranthene radical anions. The ETD process gives rise to abundant fragment ions, whose deuterium content can be measured as a function of duration of the HDX reaction in solution. No backbone protection is detected for all protein segments spanning the 25-residue long N-terminal part of the protein, which is known to lack structure in solution. At the same time, noticeable protection is evident for segments representing the structured regions of the protein. The results of this work suggest that ETD of intact protein ions is not accompanied by detectable hydrogen scrambling and can be used in tandem with HDX to probe protein conformation in solution.

Top-down proteomics on a high-field Fourier transform ion cyclotron resonance mass spectrometer.

Mass spectrometry is the tool of choice for sequencing peptides and determining the sites of posttranslational modifications; however, this bottom-up approach lacks in providing global information about the modification states of proteins including the number and types of isoforms and their stoichiometry. Recently, various techniques and mass spectrometers, such as high-field Fourier Transform Ion Cyclotron Resonance (FTICR) mass spectrometers, have been developed to study intact proteins (top-down proteomics). While the protein molecular mass and the qualitative and quantitative information about protein isoforms can be revealed by FTICR-MS analysis, their primary structure (including the identification of modifications and their exact locations in the amino acid sequence) can directly be determined using the MS/MS capability offered by the FTICR mass spectrometer. The distinct advantage of top-down methods are that modifications can be determined for a specific protein isoform rather than for peptides belonging to one or several isoforms. In this chapter, we describe different top-down proteomic approaches enabled by high-field (7, 9.4, and 12 T) FTICR mass spectrometers, and their applicability to answer biological and biomedical questions. We also describe the use of the free flow electrophoresis (FFE) to separate proteins prior to top-down mass spectrometric characterization.

Solvation of electrosprayed ions in the accumulation/collision hexapole of a hybrid Q-FTMS.

In an effort to spectroscopically determine the structures of solvated ions composed of nucleic acid bases and amino acids, methods for their gas-phase synthesis have been studied. Ions were electrosprayed and solvated in the accumulation cell of a hybrid Q-FTICR filled with methanol or water vapor at approximately 10(-2) bar. There were subsequently transferred to the FTICR cell at 10(-10) mbar. Following their isolation in the FTICR, they can be investigated by studying their unimolecular blackbody infrared radiative dissociation (BIRD) or infrared multiple photon dissociation (IRMPD) spectroscopy. The IRMPD spectra for (Ade)(2)Li(+) and (Ade)(2)Li(H(2)O)(+) are reported and compared as well as BIRD rate constants for multiply solvated and metalated adenine ions.