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Now showing items 1 - 16 of 9161

  • Melting of Hemoglobin in Native Solutions as measured by IMS-MS

    Woodall, Daniel W.   Brown, Christopher J.   Raab, Shannon A.   El-Baba, Tarick J.   Laganowsky, Arthur   Russell, David H.   Clemmer, David E.  

    Thermally induced structural transitions of the quaternary structure of the hemoglobin tetramer (human) in aqueous solution (150 mM ammonium acetate) were investigated using a variable temperature electrospray ionization (vt-ESI) technique in combination with ion mobility spectrometry (IMS) and mass spectrometry (MS) measurements. At low solution temperatures (28 to similar to 40 degrees C), a heterotetrameric (alpha(2)beta(2)) complex is the most abundant species that is observed. When the solution temperature is increased, this assembly dissociates into heterodimers (holo alpha beta forms) before ultimately forming insoluble aggregates at higher temperatures (>60 degrees C). In addition to the holo alpha beta forms, a small population of alpha beta dimers containing only a single heme ligand and having a dioxidation modification mapping to the beta subunit are observed. The oxidized heterodimers are less stable than the unmodified holo-heterodimer. The Cys(93) residue of the beta subunit is the primary site of dioxidation. The close proximity of this post translational modification to both the alpha beta subunit interface and the heme binding site suggests that this modification is coupled to the loss of the heme and decreased protein stability. Changes in the charge state and collision cross sections of these species indicate that the tetramers and dimers favor less compact structures at elevated temperatures (prior to temperatures where dissociation dominates).
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  • Proteome changes in the aging Drosophila melanogaster head

    Brown, Christopher J.   Kaufman, Thomas   Trinidad, Jonathan C.   Clemmer, David E.  

    A combination of liquid chromatography, ion mobility spectrometry, mass spectrometry, and database searching techniques were used to characterize the proteomes of four biological replicates of adult Drosophila melanogaster heads at seven time points across their lifespans. Based on the detection of tryptic peptides, the identities of 1281 proteins were determined. An estimate of the abundance of each protein, based on the three most intense peptide ions, shows that the quantified species vary in concentration over a factor of similar to 10(3). Compared to initial studies in the field of Drosophila proteomics, our current results show an eight-fold higher temporal protein coverage with increased quantitative accuracy. Across the lifespan, we observe a range of trends in the abundance of different proteins, including: an increase in abundance of proteins involved in oxidative phosphorylation, and the tricarboxylic acid cycle; a decrease in proteasomal proteins, as well as ribosomal proteins; and, many types of proteins, which remain relatively unchanged. For younger flies, proteomes are relatively similar within their age group. For older flies, proteome similarity decreases within their age group. These combined results illustrate a correlation between increasing age and decreasing proteostasis. (C) 2018 Published by Elsevier B.V.
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  • Intrinsic GTPase Activity of K-RAS Monitored by Native Mass Spectrometry

    Moghadamchargari, Zahra   Huddleston, Jamison   Shirzadeh, Mehdi   Zheng, Xueyun   Clemmer, David E.   Raushel, Frank M.   Russell, David H.   Laganows, Arthur  

    Mutations in RAS are associated with many different cancers and have been a therapeutic target for more than three decades. RAS cycles from an active to inactive state by both intrinsic and GTPase-activating protein (GAP)-stimulated hydrolysis. The activated enzyme interacts with downstream effectors, leading to tumor proliferation. Mutations in RAS associated with cancer are insensitive to GAP, and the rate of inactivation is limited to their intrinsic hydrolysis rate. Here, we use high-resolution native mass spectrometry (MS) to determine the kinetics and transition state thermodynamics of intrinsic hydrolysis for K-RAS and its oncogenic mutants. MS data reveal heterogeneity where both 2'-deoxy and 2'-hydroxy forms of GDP (guanosine diphosphate) and GTP (guanosine triphosphate) are bound to the recombinant enzyme. Intrinsic GTPase activity directly monitored by the loss in mass of K-RAS bound to GTP, which corresponds to the release of phosphate. The rates determined from MS are in direct agreement with those measured using an established solution-based assay. Our results show that the transition state thermodynamics for the intrinsic GTPase activity of K-RAS is both enthalpically and entropically unfavorable. The oncogenic mutants G12C, Q61H, and G13D unexpectedly exhibit a 2'-deoxy GTP intrinsic hydrolysis rate higher than that for GTP.
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  • "Wet" Versus "Dry" Folding of Polyproline

    Shi, Liuqing   Holliday, Alison E.   Bohrer, Brian C.   Kim, Doyong   Servage, Kelly A.   Russell, David H.   Clemmer, David E.  

    When the all-cis polyproline-I helix (PPI, favored in 1-propanol) of polyproline-13 is introduced into water, it folds into the all-trans polyproline-II (PPII) helix through at least six intermediates [Shi, L., Holliday, A.E., Shi, H., Zhu, F., Ewing, M.A., Russell, D.H., Clemmer, D.E.: Characterizing intermediates along the transition from PPI to PPII using ion mobility-mass spectrometry. J. Am. Chem. Soc. 136, 12702-12711 (2014)]. Here, we show that the solvent-free intermediates refold into the all-cis PPI helix with high (> 90%) efficiency. Moreover, in the absence of solvent, each intermediate appears to utilize the same small set of pathways observed for the solution-phase PPII -> PPI transition upon immersion of PPIIaq in 1-propanol. That folding in solution (under conditions where water is displaced by propanol) and folding in vacuo (where energy required for folding is provided by collisional activation) occur along the same pathway is remarkable. Implicit in this statement is that 1-propanol mimics a "dry" environment, similar to the gas phase. We note that intermediates with structures that are similar to PPIIaq can form PPII under the most gentle activation conditions-indicating that some transitions observed in water (i.e., "wet" folding, are accessible (albeit inefficient) in vacuo. Lastly, these "dry" folding experiments show that PPI (all cis) is favored under "dry" conditions, which underscores the role of water as the major factor promoting preference for trans proline.
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  • Cis -> Trans Isomerization of Pro(7) in Oxytocin Regulates Zn2+ Binding

    Fuller, Daniel R.   Glover, Matthew S.   Pierson, Nicholas A.   Kim, DoYong   Russell, David H.   Clemmer, David E.  

    Ion mobility/mass spectrometry techniques are employed to investigate the binding of Zn2+ to the nine-residue peptide hormone oxytocin (OT, Cys(1)-Tyr(2)-Ile(3)-Gln(4)-Asn(5)-Cys(6)-Pro(7)-Leu(8)-Gly(9)-NH2, having a disulfide bond between Cys(1) and Cys(6) residues). Zn2+ binding to OT is known to increase the affinity of OT for its receptor [Pearlmutter, A. F., Soloff, M. S.: Characterization of the metal ion requirement for oxytocin-receptor interaction in rat mammary gland membranes. J. Biol. Chem. 254, 3899-3906 (1979)]. In the absence of Zn2+, we find evidence for two primary OT conformations, which arise because the Cys(6)-Pro(7) peptide bond exists in both the trans- and cis-configurations. Upon addition of Zn2+, we determine binding constants in water of K-A =3D 1.43 +/- 0.24 and 0.42 +/- 0.12 mu M-1, for the trans- and cis-configured populations, respectively. The Zn2+ bound form of OT, having a cross section of Omega =3D 235 (2), has Pro(7) in the trans-configuration, which agrees with a prior report [Wyttenbach, T., Liu, D., Bowers, M. T.: Interactions of the hormone oxytocin with divalent metal ions. J. Am. Chem. Soc. 130, 5993-6000 (2008)], in which it was proposed that Zn2+ binds to the peptide ring and is further coordinated by interaction of the C-terminal, Pro(7)-Leu(8)-Gly(9)-NH2, tail. The present work shows that the cis-configuration of OT isomerizes to the trans-configuration upon binding Zn2+. In this way, the proline residue regulates Zn2+ binding to OT and, hence, is important in receptor binding.
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  • Configurationally-Coupled Protonation of Polyproline-7

    Shi, Liuqing   Holliday, Alison E.   Khanal, Neelam   Russell, David H.   Clemmer, David E.  

    Structure and dynamics regulate protein function, but much less is known about how biomoleculesolvent interactions affect the structure-function relationship. Even less is known about the thermodynamics of biomolecule-solvent interactions and how such interactions influence conformational entropy. When transferred from propanol into 40:60 propanol:water under acidic conditions, a remarkably slow protonation reaction coupled with the conversion of the polyproline-I helix (PPI, having all cis-configured peptide bonds) into polyproline-II (PPII, all trans) helix is observed in this work. Kinetics and equilibrium measurements as a function of temperature allow determination of the thermochemistry and insight into how proton transfer is regulated in this system. For the proton-transfer process, PPIPrOH+ + H3O+ -> PPIIPrOH/aq2+ + H2O, we determine Delta G =3D -20 +/- 19 kJ.mol(-1), Delta H =3D -75 +/- 14 kJ.mol(-1), and Delta S=3D -188 +/- 48 J.mol(-1).K-1 for the overall reaction, and values of Delta G(double dagger) =3D 91 +/- 3 kJ.mol(-1), Delta H-double dagger =3D 84 +/- 9 kJ.mol(-1), and ?S-double dagger =3D -23 +/- 31 J.mol(-1).K-1 for the transition state. For a minor process, PPIPrOH+ -> PPIIPrOH/aq+ without protonation, we determine Delta G =3D -9 +/- 20 kJ.mol(-1), Delta H =3D 64 +/- 14 kJ.mol(-1), and Delta S=3D 247 +/- 50 J.mol(-1).K-1. This thermochemistry yields Delta G =3D -10 +/- 29 kJ.mol(-1), Delta H =3D -139 +/- 20 kJ.mol(-1), and Delta S=3D -435 +/- 70 J.mol(-1).K-1 for PPIIPrOH/aq+ + H3O+ -> PPIIPrOH/aq2+ +H2O. The extraordinarily slow proton transfer appears to be an outcome of configurational coupling through a PPI-like transition state.
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    A hybrid ion mobility spectrometer includes a single-pass drift tube having an ion inlet at one end and an ion outlet at an opposite end, a multiple-pass drift tube having an ion inlet and an ion outlet each coupled to the single pass drift tube between the ion inlet and the ion outlet thereof, and a set of ion gates each controllable between an open position to pass ions therethrough and a closed position to block ions from passing therethrough. The set of ion gates may be controlled to pass at least some ions traveling through the single-pass drift tube into the multiple-pass drift tube via the ion inlet of the multiple-pass drift tube and to pass at least some ions traveling through the multiple-pass drift tube into the single-pass drift tube via the ion outlet of the multiple-pass drift tube.
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  • Oscillations of ChiralPreference in Proline Clusters.

    Holliday, Alison E.   Atlasevich, Natalya   Myung, Sunnie   Plasencia, Manolo D.   Valentine, Stephen J.   Clemmer, David E.  

    Ion mobility/mass spectrometry techniques are used tostudy the chiral preferences of small proline clusters (containing2 to 23 proline monomers) produced by electrospray ionization. Byvarying the composition of the electrospray solution from enantiomericallypure (100% lor 100% d) to racemic (50:50 l:d), it is possible to delineate which cluster sizes preferhomochiral (resolved) or heterochiral (antiresolved) compositions.The results show a remarkable oscillation in chiral preference. Singlyprotonated clusters, [xPro+H]+(where xcorresponds to the number of prolines), favor homochiralassemblies (for x= 4, 6, 11 and 12); heterochiralstructures are preferred (although the preferences are not as strong)for x= 5 and 7. Larger, doubly protonated clusters[xPro+2H]2+favor homochiral assembliesfor x= 18, 19, and 23 and heterochiral structuresfor x= 14, 16, 17, 20, 21, and 22. Some of the variationsthat are observed can be rationalized through simple structures thatwould lead to especially stable geometries. It is suggested that someantiresolved clusters, such as [22Pro+2H]2+, may be comprisedof resolved d-and l-proline domains.
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  • Analyzing a mixture of disaccharides by IMS-VUVPD-MS

    Lee, Sunyoung   Valentine, Stephen J.   Reilly, James P.   Clemmer, David E.  

    Comparative analyses utilizing collision-induced dissociation (CID) and vacuum ultraviolet photodissociation (VUVPD) for seven isobaric disaccharides have been performed in order to differentiate the linkage type and anomeric configuration of the isomers. Although an individual CID spectrum of a disaccharide ion provides information related to its structure, CID does not sufficiently differentiate mixture components due to the identical mass-to-charge values of most of the intense fragments. In contrast to the ambiguity of the CID analyses for the disaccharide mixture, VUVPD (157 nm) generates unique fragments for each disaccharide ion that are useful for distinguishing individual components from the mixture. When combined with a gas-phase ion mobility separation of the ions, the identification of each component from the mixture can be obtained. (C) 2011 Elsevier B.V. All rights reserved.
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  • Mannose7 Glycan Isomer Characterization by IMS-MS/MS Analysis

    Zhu, Feifei   Lee, Sunyoung   Valentine, Stephen J.   Reilly, James P.   Clemmer, David E.  

    The isomers of the Man(7)GlcNAc(2) glycan obtained from bovine ribonuclease B have been characterized by ion mobility spectrometry-tandem mass spectrometry (IMS-MS/MS). In these experiments, [Man7 + 2Na](2+) precursors having different mobilities are selected by ion mobility spectrometry and analyzed by MS/MS techniques in an ion trap. The fragmentation spectra obtained for various precursor ions are specific, suggesting the isolation or enrichment of different glycan isomers. One fragment ion with a mass-to-charge ratio (m/z) of 903.8 is found to correspond to the loss of an internal mannose residue of a specific isomer. Extracted fragment ion drift time distributions (XFIDTDs) yield distinctive precursor ion drift time profiles indicating the existence of four separate isomers as proposed previously.
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  • Biomolecular condensation via ultraviolet excitation in vacuo

    Lee, Sunyoung   Julian, Ryan R.   Valentine, Stephen J.   Reilly, James P.   Clemmer, David E.  

    Recently, we reported that 157 nm vacuum ultraviolet irradiation (VUV) of proton-bound peptide dimers trapped in a vacuum, results in the elimination of water and formation of a peptide bond [J. Am. Chem. Soc. 133 (2011) 15834-15837]. Here, we further explore the ability to form a covalent bond between biomolecular ions with photoexcitation. Photoexcitation of long-lived charge-bound complexes appears to be a general phenomenon, resulting in the loss of water and the formation of covalent bonds between many types of molecules. Several examples are described, including: the linear coupling of amino acid chains that produce octapeptides from tetrapeptide complexes: inter-molecular cross-linking of amino acid side chains, and glycosidic bond formation between disaccharide complexes. Simple mechanisms for each case are proposed. (c) 2012 Published by Elsevier B.V.
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  • Chirality and Packing in Small Proline Clusters

    Atlasevich, Natalya   Holliday, Alison E.   Valentine, Stephen J.   Clemmer, David E.  

    The chiral composition of amino acid clusters may be related to the origin of chirality in biological systems. Here, we use ion mobility/mass spectrometry techniques to investigate the gas phase structures of singly charged proline clusters containing two to six monomers. Using deuterated L-proline (L-D7) and different electrospray solution compositions varying from enantiopure (50:50 L:LD7) to racemic (50:50 LD7:D), it is possible to study collision cross sections of L-, D-, and mixed xL:xD-proline clusters (where x refers to the number of monomers). These results show that [2Pro+H](+) and [3Pro+H](+) clusters, previously shown (Holliday et al. J. Phys. Chem. A 2012., DOI: 10.1021/jp302677n) to have a very small heterochiral preference, have similar collision cross sections for homochiral and heterochiral proline assemblies. The [4Pro+H](+) and [6Pro+H](+) clusters that exhibit homochiral preference have smaller collision cross sections for homochiral clusters and larger collision cross sections for heterochiral clusters. The [SPro+H](+) cluster with heterochiral preference has a smaller collision cross section for its heterochiral compositions than for its homochiral compositions. These results suggest that the packing efficiency of subunits within each cluster influences the stability and prevalence of proline multimers as either homochiral or mixed L- and 1)clusters.
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  • Characterizing the Conformationome:Toward a Structural Understanding of the Proteome

    Clemmer, David E.   Russell, David H.   Williams, Evan R.  

    While non-native protein conformations such as folding intermediates are rarely observed in solution such species are often stabilized as gaseous ions during electrospray ionization for mass spectrometry. This opens the possibility of large scale efforts to capture information about many non-native structures such as folding intermediates or malformed conformations having deleterious effects: studies of the conformationome.
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  • Overtone Mobility Spectrometry: Part 4. OMS-OMS Analyses of Complex Mixtures

    Kurulugama, Ruwan T.   Nachtigall, Fabiane M.   Valentine, Stephen J.   Clemmer, David E.  

    A new, two-dimensional overtone mobility spectrometry (OMS-OMS) instrument is described for the analysis of complex peptide mixtures. OMS separations are based on the differences in mobilities of ions in the gas phase. The method utilizes multiple drift regions with modulated drift fields such that only ions with appropriate mobilities are transmitted to the detector. Here we describe a hybrid OMS-OMS combination that utilizes two independently operated OMS regions that are separated by an ion activation region. Mobility-selected ions from the first OMS region are exposed to energizing collisions and may undergo structural transitions before entering the second OMS region. This method generates additional peak capacity and allows for higher selectivity compared with the one-dimensional OMS method. We demonstrate the approach using a three-protein tryptic digest spiked with the peptide Substance P. The [M+3H](3+) ion from Substance P can be completely isolated from other components in this complex mixture prior to introduction into the mass spectrometer.
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  • An appreciation

    Rodgers, Mary T.   Clemmer, David E.  

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    Apparatus, systems, and methods for determining the mass of one or more ions at high pressure are disclosed. The subject devices may operate by and the subject methods may include receiving one or more drift time signals indicative of the time required by a first ion or group of ions to traverse a drift tube operating at or near atmospheric pressure, receiving one or more drift time signals indicative of the time required by a second ion or group of ions to traverse the drift tube, wherein the second ion or group of ions have similar collision cross-sections but different ion masses from the first ion or group of ions, and calculating a mass measurement for at least one of the first ion or group of ions or the second ion or groups of ions which is substantially independent of the collision cross-sections.
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