Exposure to inorganic arsenic, a ubiquitous environmental toxic metalloid, leads to carcinogenesis. However, the mechanism is unknown. Several studies have shown that inorganic arsenic exposure ...alters specific gene expression patterns, possibly through alterations in chromatin structure. While most studies on understanding the mechanism of chromatin-mediated gene regulation have focused on histone post-translational modifications, the role of histone variants remains largely unknown. Incorporation of histone variants alters the functional properties of chromatin. To understand the global dynamics of chromatin structure and function in arsenic-mediated carcinogenesis, analysis of the histone variants incorporated into the nucleosome and their covalent modifications is required. Here we report the first global mass spectrometric analysis of histone H2B variants as cells undergo arsenic-mediated epithelial to mesenchymal transition. We used electron capture dissociation-based top-down tandem mass spectrometry analysis validated with quantitative reverse transcription real-time polymerase chain reaction to identify changes in the expression levels of H2B variants in inorganic arsenic-mediated epithelial-mesenchymal transition. We identified changes in the expression levels of specific histone H2B variants in two cell types, which are dependent on dose and length of exposure of inorganic arsenic. In particular, we found increases in H2B variants H2B1H/1K/1C/1J/1O and H2B2E/2F, and significant decreases in H2B1N/1D/1B as cells undergo inorganic arsenic-mediated epithelial-mesenchymal transition. The analysis of these histone variants provides a first step toward an understanding of the functional significance of the diversity of histone structures, especially in inorganic arsenic-mediated gene expression and carcinogenesis.
Molecular formulas have been assigned for 4626 individual Suwannee River fulvic acids based on accurate mass measurements from ions generated by electrospray ionization and observed by ...ultrahigh-resolution Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS). Formula assignments were possible because of the mass accuracy of FTICR MS at high field (9.4 T) and the regular mass spacing patterns found in fulvic acid mixtures. Sorting the 4626 individually observed ions according to Kendrick mass defect and nominal mass series (z* score) revealed that all could be assigned to 1 of 266 distinct homologous series that differ in oxygen content and double bond equivalence. Tandem mass spectrometry based on infrared multiphoton dissociation identified labile fragments of fulvic acid molecules, whose chemical formulas led to plausible structures consistent with degraded lignin as a source of Suwannee River fulvic acids.
COPII vesicles transport proteins from the endoplasmic reticulum to the Golgi apparatus. Previous COPII-cage cryo-EM structures lacked the resolution necessary to determine the residues of Sec13 and ...Sec31 that mediate assembly and flexibility of the COPII cage. Here we present a 12-Å structure of the human COPII cage, where the tertiary structure of Sec13 and Sec31 is clearly identifiable. We employ this structure and a homology model of the Sec13-Sec31 complex to create a reliable pseudoatomic model of the COPII cage. We combined this model with hydrogen/deuterium-exchange MS analysis to characterize four distinct contact regions at the vertices of the COPII cage. Furthermore, we found that the two-fold symmetry of the Sec31 dimeric region in Sec13-Sec31 is broken upon cage formation and that the resulting hinge is essential to form the proper edge geometry in COPII cages.
Fourier‐transform ion cyclotron resonance mass spectrometry (MS) has demonstrated potential to revolutionize the fields of limnology and chemical oceanography by identifying the individual molecular ...components of organic matter in natural waters. The use of MS for this purpose is made possible by the electrospray technique which successfully ionizes polar, nonvolatile organic molecules. Another recently developed ion source, atmospheric pressure photoionization (APPI), extends MS capabilities to less polar molecules. This article presents early results on the application of APPI MS to natural organic matter. We compare APPI MS and electrospray MS data for dissolved organic matter from Lake Drummond (Virginia, USA). Collectively, electrospray and APPI MS identify more than 6000 molecular species to which we assign unique molecular formulas. Fewer than 1000 molecular species are common to both electrospray and APPI mass spectra, indicating that the techniques are highly complementary in the types of molecules they ionize. Access to a broad range of molecules provided by combining APPI and electrospray has prompted a qualitative analysis. The goal is to assess the extent to which molecular MS data correspond with elemental (CHNOS) and structural characteristics determined by combustion elemental analyses and 13C nuclear magnetic resonance (NMR). Because the data obtained by these different methods are not directly comparable, we propose a novel data analysis procedure that facilitates their comparison. The bulk elemental composition calculated from electrospray MS data are in close agreement (± 15%) with values determined by combustion elemental analysis. APPI and electrospray MS detect protein contributions in agreement with 13C NMR (6 wt %) but underestimate carbohydrates relative to 13C NMR. Nevertheless, MS results agree with NMR on the relative proportions of noncarbohydrate compounds in the organic matter: lignins > lipids > peptides. Finally, we use a molecular mixing model to simulate a 13C NMR spectrum from the MS datasets. The correspondence of the simulated and measured 13C NMR signals (74%) suggests that, collectively, the molecular species identified by APPI and electrospray MS comprise a large portion of the organic matter in Lake Drummond. These results add credibility to electrospray and APPI MS in limnology and oceanography applications, but further characterization of ion source behavior is fundamental to the accurate interpretation of MS data.
Mass spectrometry is well-suited for complex mixture analysis, because unlike other types of spectroscopy, the number of mass spectral peaks per analyte is of order one. Here, we extend significantly ...the upper limit for the number of chemically distinct components resolved and identified in a single step. Specifically, electrospray selectively ionizes only the basic compounds (i.e., a small fraction of the entire chemical composition) in a sample of South American crude oil. Nevertheless, their positive ion Fourier transform ion cyclotron resonance mass spectrum (average mass resolving power of ∼350 000 from 225 to 1000 Da) contains more than 11 100 resolved peaks, of which >75% may be assigned to a unique elemental composition (C c H h O o N n S s ). Mass scale expansion and graphical representations reveal increased heteroatom diversity, aromatic rings, and alkyl substitution with increased mass. These results set a new standard and illustrate the potential of high-resolution mass spectrometry for analysis of compositionally complex chemical mixtures.
Ultrahigh-resolution electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry has resolved and identified the elemental compositions of over 10 000 organic constituents of ...coal and petroleum crude oil. A plot of Kendrick mass defect versus Kendrick nominal mass sorts compounds into homologous series according to compound class (i.e., numbers of N, O, and S heteroatoms), type (number of rings plus double bonds), and degree of alkylation (number of CH2 groups), to yield unique elemental assignments from ultrahigh-resolution mass measurements in the 200−900 Da range. Interpretation of such a vast compilation requires a simple (preferably graphical) means to differentiate between complex organic mixtures of different origin or processing. In an extension of the recently revived van Krevelen plot, each elemental composition is projected onto two or three axes according to its H/C, O/C, and/or N/C atomic ratios. The H/C ratio separates compounds according to degree of saturation, whereas O/C or N/C ratios separate according to O and N classes. We show that the three-dimensional van Krevelen diagram can completely separate different classes in pyridine-extracted coal or petroleum samples and can also graphically distinguish fossil fuels according to their nature (coal vs petroleum), maturation (coals of different rank), and processing (the same coal at two stages of liquefaction). The van Krevelen diagram thus appears well suited to amplifying and exposing compositional differences within and between complex organic mixtures.
Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS) provides the highest mass resolving power and mass measurement accuracy for unambiguous identification of biomolecules. ...Previously, the highest-mass protein for which FTICR unit mass resolution had been obtained was 115 kDa at 7 T. Here, we present baseline resolution for an intact 147.7 kDa monoclonal antibody (mAb), by prior dissociation of noncovalent adducts, optimization of detected total ion number, and optimization of ICR cell parameters to minimize space charge shifts, peak coalescence, and destructive ion cloud Coulombic interactions. The resultant long ICR transient lifetime (as high as 20 s) results in magnitude-mode mass resolving power of ∼420 000 at m/z 2 593 for the 57+ charge state (the highest mass for which baseline unit mass resolution has been achieved), auguring for future characterization of even larger intact proteins and protein complexes by FTICR MS. We also demonstrate up to 80% higher resolving power by phase correction to yield an absorption-mode mass spectrum.
Asphaltenes are typically defined by their solubility in benzene and insolubility in pentane or heptane. They are believed to exist in petroleum crude oil as a colloidal suspension, stabilized by ...surface-adsorbed resins. Their normal equilibrium under reservoir conditions may be disrupted during production by pressure reduction, crude oil chemical composition changes, introduction of miscible gases and liquids, and mixing with diluents and other oils, as well as by acid stimulation, hot oiling, and other oilfield operations. Electrospray ionization preferentially ionizes polar N-, S-, and O-containing compounds, and its combination with ultrahigh-resolution Fourier transform ion cyclotron resonance mass spectrometry makes a powerful tool for the compositional analysis of petroleum-derived materials such as asphaltenes. In this work, we compare the compositional differences between heptane-precipitated asphaltenes and asphaltenes collected by live oil depressurization. Negative- and positive-ion electrospray yield the acidic and basic species, respectively. We find that the heptane-precipitated asphaltenes contain higher double bond equivalents (number of rings plus double bonds) compared to the asphaltenes induced by pressure drop. On the other hand, the pressure-drop product exhibits a higher abundance of species containing sulfur. Thus, the solubility criterion for asphaltenes defines a significantly different chemical composition than the (more field-relevant) pressure-drop criterion.
Amide hydrogen/deuterium exchange is a powerful biophysical technique for probing changes in protein dynamics induced by ligand interaction. The inherent low throughput of the technology has limited ...its impact on drug screening and lead optimization. Automation increases the throughput of H/D exchange to make it compatible with drug discovery efforts. Here we describe the first fully automated H/D exchange system that provides highly reproducible H/D exchange kinetics from 130 ms to 24 h. Throughput is maximized by parallel sample processing, and the system can run H/D exchange assays in triplicate without user intervention. We demonstrate the utility of this system to differentiate structural perturbations in the ligand-binding domain (LBD) of the nuclear receptor PPARγ induced upon binding a full agonist and a partial agonist. PPARγ is the target of glitazones, drugs used for treatment of insulin resistance associated with type II diabetes. Recently it has been shown that partial agonists of PPARγ have insulin sensitization properties while lacking several adverse effects associated with full agonist drugs. To further examine the mechanism of partial agonist activation of PPARγ, we extended our studies to the analysis of ligand interactions with the heterodimeric complex of PPARγ/RXRα LBDs. To facilitate analysis of H/D exchange of large protein complexes, we performed the experiment with a 14.5-T Fourier transform ion cyclotron resonance mass spectrometer capable of measuring mass with accuracy in the ppb range.
The question of how dispersed mutations in one protein engender the same gain-of-function phenotype is of great interest. Here we focus on mutations in glycyl-tRNA synthetase (GlyRS) that cause an ...axonal form of Charcot–Marie–Tooth (CMT) diseases, the most common hereditary peripheral neuropathies. Because the disease phenotype is dominant, and not correlated with defects in the role of GlyRS in protein synthesis, the mutant proteins are considered to be neomorphs that gain new functions from altered protein structure. Given that previous crystal structures showed little conformational difference between dimeric wild-type and CMT-causing mutant GlyRSs, the mutant proteins were investigated in solution by hydrogen-deuterium exchange (monitored by mass spectrometry) and small-angle X-ray scattering to uncover structural changes that could be suppressed by crystal packing interactions. Significantly, each of five spatially dispersed mutations induced the same conformational opening of a consensus area that is mostly buried in the wild-type protein. The identified neomorphic surface is thus a candidate for making CMT-associated pathological interactions, and a target for disease correction. Additional result showed that a helix-turn-helix WHEP domain that was appended to GlyRS in metazoans can regulate the neomorphic structural change, and that the gain of function of the CMT mutants might be due to the loss of function of the WHEP domain as a regulator. Overall, the results demonstrate how spatially dispersed and seemingly unrelated mutations can perpetrate the same localized effect on a protein.
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