Resolution and structural transitions of elongated states of ubiquitin

Electrospray ionization, combined with two-dimensional ion mobility spectrometry and mass spectrometry, is used to produce, select, and activate distributions of elongated ions, [M ϩ 11H] 11ϩ to [M ϩ 13H] 13ϩ , of ubiquitin. The analysis makes it possible to examine state-to-state transitions for structural types, and transition diagrams associated with the efficiencies of structural changes are presented. The ϩ11 and ϩ12 charge states can form four resolvable states while only one state is formed for [M ϩ 13H] 13ϩ . Some conformations, which appear to belong to the same family based on mobility analysis of different charge states, undergo similar transitions, others do not. Activation of ions that exist in low-abundance conformations, having mobilities that fall in between sharp peaks associated with higher abundances species, shows that the low-abundance forms undergo efficient (ϳ90 to 100%) conversion into states associated with well-defined peaks. This efficiency is significantly higher than the ϳ10 to 60% efficiency of transitions of structures associated with well-defined peaks. The formation of sharp features from a range of low-intensity species with different cross sections indicates that large regions of conformation space must be unfavorable or inaccessible in the gas phase. These results are compared with several previous IMS measurements of this system as well as information about gas-phase structure provided by other techniques. Studies of solvent-free proteins and peptides are important because of both fundamental and practical considerations. In the absence of solvation shells (or with minimal solvent), it is possible to extract kinetic and thermodynamic benchmarks about the formation of specific types of folds and tease out the influence of solvent-molecule and intramolecular factors in establishing conformation [2] A number of groups have worked to combine ion mobility spectrometry (IMS) and MS with the aim of using differences in ion mobility to separate components of a mixture that would not be resolved by MS alone [21, 22]. The mobility of a macromolecular ion through a buffer gas depends on its charge and shape (average collision cross section with the buffer gas, ⍀). Recently, we have ex- tended a hybrid IMS/MS instrument to include additional IMS dimensions leading to IMS-IMS/MS and IMS-IMS-IMS/MS instrument designs As one develops new IMS techniques (as well as other methods) it is important to revisit model systems, where some fundamental understanding of the nature of the system exists. In the present paper, we focus on the ϩ11 to ϩ13 charge states of ubiquitin ions produced by standard ESI conditions. Ubiquitin is a small, 76 amino acid protein that, under most ESI source conditions, favors the ϩ5 to ϩ13 charge states [27

Screening and sequencing of sialylated glycosphingolipids in human glioblastoma by ion mobility mass spectrometry

High performance ion mobility separation mass spectrometry (IMS MS) was thoroughly optimized to allow the discovery of glioblastoma multiforme (GBM)-specific structures and the assessment of their roles as tumor markers or possible associated antigens. Ganglioside (GG) separation by IMS according to the charge state, carbohydrate chain length, degree of sialylation and ceramide composition, led to the identification of no less than 160 distinct components [1], which represents 3 folds the number of structures identified before. The detected GGs and asialo-GGs were found characterized by a high heterogeneity in their ceramide and glycan compositions, encompassing up five Neu5Ac residues. The tumor was found dominated in equal and high proportions by GD3 and GT1 forms, with a particular incidence of C24:1 fatty acids in the ceramide

Mapping the Human Plasma Proteome by SCX-LC-IMS-MS

The advent of on-line multidimensional liquid chromatography-mass spectrometry has significantly impacted proteomic analyses of complex biological fluids such as plasma. However, there is general agreement that additional advances to enhance the peak capacity of such platforms are required to enhance the accuracy and coverage of proteome maps of such fluids. Here, we describe the combination of strong-cation-exchange and reversed-phase liquid chromatographies with ion mobility and mass spectrometry as a means of characterizing the complex mixture of proteins associated with the human plasma proteome. The increase in separation capacity associated with inclusion of the ion mobility separation leads to generation of one of the most extensive proteome maps to date. The map is generated by analyzing plasma samples of five healthy humans; we report a preliminary identification of 9087 proteins from 37,842 unique peptide assignments. An analysis of expected false-positive rates leads to a high-confidence identification of 2928 proteins. The results are catalogued in a fashion that includes positions and intensities of assigned features observed in the datasets as well as pertinent identification information such as protein accession number, mass, and homology score/confidence indicators. Comparisons of the assigned features reported here with other datasets shows substantial agreement with respect to the first several hundred entries; there is far less agreement associated with detection of lower abundance components

Overtone Mobility Spectrometry: Part 2. Theoretical Considerations of Resolving Power

The transport of ions through multiple drift regions is modeled to develop an equation that is useful for an understanding of the resolving power of an overtone mobility spectrometry (OMS) technique. It is found that resolving power is influenced by a number of experimental variables, including those that define ion mobility spectrometry (IMS) resolving power: drift field (E), drift region length (L), and buffer gas temperature (T). However, unlike IMS, the resolving power of OMS is also influenced by the number of drift regions (n), harmonic frequency value (m), and the phase number (⌽) of the applied drift field. The OMS resolving power dependence upon the new OMS variables (n, m, and ⌽) scales differently than the square root dependence of the E, L, and T variables in IMS. The results provide insight about optimal instrumental design and operation. (J Am Soc Mass Spectrom 2009, 20, 738 -750) © 2009 American Society for Mass Spectrometry W hen a pulse of ions is injected into a buffer gas, different species separate under the influence of an electric field because of differences in their mobilities through the buffer gas Of note is the dependence of resolving power on the square root of the various parameters. This relationship imposes limits on the ultimate instrument performance. For example, doubling L does not double the resolving power; rather, a 2-fold increase in L (holding T and E constant) results in only ϳ40% increase in resolving power. In the present paper, we report modeling studies of ion transport through multiple drift regions to which the drift fields are applied at varying frequencies, the experimental setup used in overtone mobility spectrometry (OMS). The understanding that is gained from modeling allows us to develop a simple equation that can be used to estimate the OMS resolving power (R OMS ). The equation describing the OMS resolving power accounts for a number of geometrical OMS device configurations as well as those parameters used to define R IMS . However, these studies indicate that variation of the parameters which define R IMS (E, L, and T) have only a limited impact on the R OMS . Instead, the factors having the greatest influence on R OMS appear to be the number of phases for the system (i.e., the number of unique drift field application settings as well as the number of drift regions in a complete ion transmission/ elimination cycle, see below for complete description), the overall number of ion drift regions, and the drift field setting frequency (overtone number). A surprising result is the unit proportionality relationship between R OMS and the number of drift regions (in effect L) as well as the frequency suggesting the ability to garner much improved instrument performance (with respect to resolution) for proportionate changes when compared with IMS techniques. It is important to note that the comparisons to R IMS (with respect to L) described here are based on the use of a constant drift field. Equation 1 can be rewritten such that the product of L and E is denoted as the drift voltage (V). Extensive work has shown that increased resolving power can be obtained by optimizing V and Address reprint requests to Dr

Reaction of Zn^+ with NO_2. The gas-phase thermochemistry of ZnO

The homolytic bond dissociation energies of ZnO and ZnO^+ have been determined by using guided ion‐beam mass spectrometry to measure the kinetic‐energy dependence of the endothermic reactions of Zn^+ with nitrogen dioxide. The data are interpreted to yield the bond energy for ZnO, D^0_0=1.61±0.04 eV, a value considerably lower than previous experimental values, but in much better agreement with theoretical calculations. We also obtain D^0_0(ZnO^+)=1.67±0.05 eV, in good agreement with previous results. Other thermochemistry derived in this study is D^0_0(Zn^+–NO)=0.79±0.10 eV and the ionization energies, IE(ZnO)=9.34±0.02 eV and IE(NO_2)=9.57±0.04 eV

Characterization of hybrid chondroitin/dermatan sulfate octasaccharide domains in human brain by ion mobility tandem mass spectrometry

We report here on the introduction of a rapid, highly sensitive and reliable approach in a single run, based on ion mobility separation (IMS), high resolution and tandem MS (MS/MS) by collision-induced dissociation (CID) for compositional and structural elucidation of neural chondroitin sulfate (CS) and dermatan sulfate (DS) domains, which implies the determination of the epimerization and the sulfation code of regular and irregular structures. By IMS MS and CID MS/MS, we were able to characterize in details CS/DS octasaccharides from brain obtained after CS/DS chain depolymerization by chondroitin B lyase and to detect sequences that were never found before in the octasaccharide domains of the investigated CS/DS brain fraction

Adsorption Isotherms of Hydrogen: The Role of Thermal Fluctuations

It is shown that experimentally obtained isotherms of adsorption on solid substrates may be completely reconciled with Lifshitz theory when thermal fluctuations are taken into account. This is achieved within the framework of a solid-on-solid model which is solved numerically. Analysis of the fluctuation contributions observed for hydrogen adsorption onto gold substrates allows to determine the surface tension of the free hydrogen film as a function of film thickness. It is found to decrease sharply for film thicknesses below seven atomic layers.Comment: RevTeX manuscript (3 pages output), 3 figure

Profiling and structural characterization by mass spectrometry of region-specific gangliosides in brain

Gangliosides (GGs), a particular class of glycosphingolipids ubiquitously found in tissues and body fluids, exhibit the highest expression in the central nervous system, especially in brain. GGs are involved in crucial processes, such as neurogenesis, synaptogenesis, synaptic transmission, cell adhesion, growth and proliferation. For these reasons, efforts are constantly invested into development and refinement of specific methods for GG analysis. We have recently shown that ion mobility separation (IMS) mass spectrometry (MS) has the capability to provide consistent compositional and structural information on GGs at high sensitivity, resolution and mass accuracy. In the present study we have implemented IMS MS for the first time in the study of a highly complex native GG mixture extracted and purified from a normal fetal hippocampus in the 17th gestational week (denoted FH17). The combination of electrospray ionization, ion mobility separation and high resolution mass spectrometry in the negative ionization mode enhanced ganglioside separation based not just on the m/z value, but also on the charge state, the carbohydrate chain length and the degree of sialylation. In the generated driftscope plot (drift time versus m/z), 131 distinct gangliosides characterized by high variability of the oligosaccharide core and diversity of the ceramide moiety were identified with an average mass accuracy of 12.3 ppm. As compared to previous studies where no separation techniques prior to MS were applied, IMS MS technique has not just generated valuable novel information on the GG pattern characteristic for hippocampus in early developmental stage, but also provided data related to the GG molecular involvement in the synaptic functions by the discovery of 25 novel structures modified by CH3COO- . By applying IMS in conjunction with collision induced dissociation (CID) tandem MS (MS/MS), novel GG species modified by CH3COOattachment, discovered here for the first time, were sequenced and structurally investigated in details. The present findings, based on IMS MS, provide a more reliable insight into the expression and role of gangliosides in human hippocampus, with a particular emphasis on their cholinergic activity at this level

Ion mobility mass spectrometry of gangliosides in human brain in health and disease

Gangliosides (GGs), a class of glycosphingolipids, are important biomarkers in early diagnosis of CNS pathologies, being in the focus of our research as potential therapeutic targets [1]. A series of neuropsychiatric disorders are characterized by amnesia and disorientation caused by hippocampal atrophy and diminished cholinergic activity. Based on ion mobility separation mass spectrometry (IMS MS) capability for a reliable glycopattern determination, and the occurrence of neuropsychiatric disorders [2,3], we report here on the improvement of novel and high performance IMS MS method for assessing the GG profile in a highly complex mixture extracted from an adult healthy brain region. The IMS separation of GGs based on charge state, carbohydrate chain length and degree of sialylation led to the detection and identification of over 100 species, the larger number of GGs ever reported before in this particular brain region. Moreover, the obtained data supports the concept of GGs cholinergic activity. Furthermore, by applying IMS MS/MS, novel GG species were structurally investigated in details