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

Tautomerization of H⁺KPGG: Entropic Consequences of Strong Hydrogen-Bond Networks in Peptides

Ion mobility spectrometry-mass spectrometry and quantum chemical calculations are used to determine the structures and stabilities of the singly protonated peptide H+KPGG. The two peaks making up the IMS distribution are shown to be tautomers differing by the location of the extra proton on either the lysine side chain or the N-terminus. The lysine-protonated tautomer is strongly preferred entropically while being disfavored in terms of the electronic energy and enthalpy. This relationship is shown, through comparison of all low-lying conformers of both tautomers, to be related to the strong hydrogen-bond network of the N-terminally protonated tautomer. A general relationship is demonstrated wherein stronger cross-peptide hydrogen-bond networks result in entropically disfavored conformers. Further effects of the H+KPGG hydrogen-bond network are probed by computationally examining singly and doubly methylated analogues. These results demonstrate the importance of the entropic consequences of hydrogen bonds to peptide stability as well as techniques for perturbing the hydrogen-bond network and folding preferences of peptides via minimal chemical modification

Achieving favourable customer outcomes through employee deviance

This study advances current knowledge by examining how employee deviance and customer participation during a single employee-customer exchange generate favourable customer responses. This work bridges the employee deviance stream with the service encounter literature and illustrates the importance of equity theory in deviant service exchanges between customers and employees. Moreover, results add to the ongoing debate on service nepotism by canvassing the consequences from the customer’s active participation in deviant exchanges which appears to enhance customer perceptions of the exchange. A 3x2 between-subjects experimental design was adopted which manipulates three types of pro-customer deviance along with customer’s participation (or not) to the exchange. The dependent variables capture three types of perceived customer justice (cognitive outcomes) and customer’s affective state (affective outcome). Findings illustrate that customers approve employees’ deviance for their own benefit while also indicate favourable outcomes from deviant exchanges with employees such as higher perceived justice and a more positive affective state. The article concludes with a discussion of the theoretical and managerial implications, limitations and research directions that emerge from this study

Native bluegill influence the foraging and aggressive behavior of invasive mosquitofish

Two fish species that are common invaders of aquatic ecosystems world-wide are Gambusia affinis and G. holbrooki, commonly known as mosquitofish. In North America, introduced G. affinis are thought to have contributed to the population decline of several native fish species. Sunfish (family Centrarchidae) naturally occur across much of North American, thus mosquitofish and sunfish are likely to come into contact and interact more frequently as mosquitofish spread. However, the nature of this interaction is not well known. We used a lab experiment to explore whether and how the aggressive and foraging behaviors of G. affinis might be influenced by a representative and ubiquitous native centrarchid (Lepomis macrochirus; bluegill sunfish), a species with juveniles that inhabit littoral habitats also preferred by mosquitofish. The experiment partnered an individual male or female mosquitofish (focal fish) with a juvenile bluegill, or a same- or opposite-sex conspecific, filmed these one-to-one interactions, and quantified foraging and aggressive actions for the focal mosquitofish. We found that juvenile bluegill affect foraging in male mosquitofish, resulting in lower percent of handling attempts and handling time in which the male consumed a food item. The presence of juvenile bluegill also led to a reduction in the number of aggressive acts by mosquitofish compared to aggression levels when focal mosquitofish were with conspecifics. In nature, when mosquitofish encounter juvenile bluegill in littoral habitats, our results suggest that the foraging and aggressive behaviors of mosquitofish will be modified, especially for males. This mechanism may influence the rate or geographic extent of the spread of mosquitofish into North American waterbodies

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