Analyzing the higher order structure of proteins with conformer-selective ultraviolet photodissociation

The top-down approach in protein sequencing requires simple methods in which the analyte can be readily dissociated at every position along the backbone. In this context, ultraviolet photodissociation (UVPD) recently emerged as a promising tool because, in contrast to slow heating techniques such as collision induced dissociation (CID), the absorption of UV light is followed by a rather statistically distributed cleavage of backbone bonds. As a result, nearly complete sequence coverage can be obtained. It is well-known, however, that gas-phase proteins can adopt a variety of different, sometimes coexisting conformations and the influence of this structural diversity on the UVPD fragmentation behavior is not clear. Using ion mobility-UVPD-mass spectrometry we recently showed that UVPD is sensitive to the higher order structure of gas-phase proteins. In particular, the cis/trans isomerization of certain proline peptide bonds was shown to significantly influence the UVPD fragmentation pattern of two extended conformers of 11+ ubiquitin. Building on these results, we here provide conformer-selective UVPD data for 7+ ubiquitin ions, which are known to be present in a much more diverse and wider ensemble of different structures, ranging from very compact to highly extended species. Our data show that certain conformers fall into groups with similar UVPD fragmentation pattern. Surprisingly, however, the conformers within each group can differ tremendously in their collision cross section. This indicates that the multiple coexisting conformations typically observed for 7+ ubiquitin are caused by a few, not easily inter-convertible, subpopulations

Repulsion-Phase Linkage Analysis of Tetraploid Creeping Bentgrass (Agrostis Stolonifera L.)

Creeping bentgrass is a cool-season grass species primarily used on golf course greens, tees, and fairways because of its tolerance of low mowing heights and recuperative ability. Creeping bentgrass is tetraploid and outcrossing and has been characterized as having bivalent chromosome pairing through cytogenetic analysis (Jones, 1956) and disomic inheritance based on the inheritance of isozyme markers (Warnke et al., 1998). The objective of this experiment was to evaluate the extent of repulsion-phase linkages of single dose AFLP type markers in a creeping bentgrass mapping population and to infer chromosome pairing behaviour based on the ratio of coupling to repulsion-phase linkages

Photodissociation of Conformer-Selected Ubiquitin Ions Reveals Site-Specific Cis/Trans Isomerization of Proline Peptide Bonds

Ultraviolet photodissociation (UVPD) of gas-phase proteins has attracted increased attention in recent years. This growing interest is largely based on the fact that, in contrast to slow heating techniques such as collision induced dissociation (CID), the cleavage propensity after absorption of UV light is distributed over the entire protein sequence, which can lead to a very high sequence coverage as required in typical top-down proteomics applications. However, in the gas phase, proteins can adopt a multitude of distinct and sometimes coexisting conformations, and it is not clear how this three-dimensional structure affects the UVPD fragmentation behavior. Using ion mobility–UVPD–mass spectrometry in conjunction with molecular dynamics simulations, we provide the first experimental evidence that UVPD is sensitive to the higher order structure of gas-phase proteins. Distinct UVPD spectra were obtained for different extended conformations of 11+ ubiquitin ions. Assignment of the fragments showed that the majority of differences arise from cis/trans isomerization of one particular proline peptide bond. Seen from a broader perspective, these data highlight the potential of UVPD to be used for the structural analysis of proteins in the gas phas

From Compact to String—The Role of Secondary and Tertiary Structure in Charge-Induced Unzipping of Gas-Phase Proteins

In the gas phase, protein ions can adopt a broad range of structures, which have been investigated extensively in the past using ion mobility-mass spectrometry (IM-MS)-based methods. Compact ions with low number of charges undergo a Coulomb-driven transition to partially folded species when the charge increases, and finally form extended structures with presumably little or no defined structure when the charge state is high. However, with respect to the secondary structure, IM-MS methods are essentially blind. Infrared (IR) spectroscopy, on the other hand, is sensitive to such structural details and there is increasing evidence that helices as well as β-sheet-like structures can exist in the gas phase, especially for ions in low charge states. Very recently, we showed that also the fully extended form of highly charged protein ions can adopt a distinct type of secondary structure that features a characteristic C5-type hydrogen bond pattern. Here we use a combination of IM-MS and IR spectroscopy to further investigate the influence of the initial, native conformation on the formation of these structures. Our results indicate that when intramolecular Coulomb-repulsion is large enough to overcome the stabilization energies of the genuine secondary structure, all proteins, regardless of their sequence or native conformation, form C5-type hydrogen bond structures. Furthermore, our results suggest that in highly charged proteins the positioning of charges along the sequence is only marginally influenced by the basicity of individual residues

Achlioptas processes are not always self-averaging

We consider a class of percolation models, called Achlioptas processes, discussed in [Science 323, 1453 (2009)] and [Science 333, 322 (2011)]. For these the evolution of the order parameter (the rescaled size of the largest connected component) has been the main focus of research in recent years. We show that, in striking contrast to `classical' models, self-averaging is not a universal feature of these new percolation models: there are natural Achlioptas processes whose order parameter has random fluctuations that do not disappear in the thermodynamic limit.Comment: 4 pages, 3 figures. Revised and expanded. Title change

Online monitoring the isomerization of an azobenzene-based dendritic bolaamphiphile using ion mobility-mass spectrometry

Ion mobility-mass spectrometry was used to obtain detailed information about the kinetics of the light-induced cis/trans isomerization process of a new supramolecular azobenzene-based bolaamphiphile. Further experiments revealed that the investigated light-induced structural transition dramatically influences the aggregation behaviour of the molecule

Consistency of QTL for Dollar Spot Resistance Between Greenhouse and Field Inoculations, Multiple Locations, and Different Population Sizes in Creeping Bentgrass

Dollar spot caused by Sclerotinia homoeocarpa F. T. Bennett is the most economically important turf disease in North America. Previous work indicated differences among cultivars in their susceptibility to dollar spot (Bonos et al., 2003). Studies have indicated that dollar spot resistance might be quantitatively inherited (Bonos et al., 2003) but the number, location and effect of genomic regions conferring resistance is still not known. Therefore the objective of this research is to understand the effect of population size, inoculation assays, and field locations on QTL for dollar spot resistance in creeping bentgrass

Native like helices in a specially designed β peptide in the gas phase

In the natural peptides, helices are stabilized by hydrogen bonds that point backward along the sequence direction. Until now, there is only little evidence for the existence of analogous structures in oligomers of conformationally unrestricted β amino acids. We specifically designed the β peptide Ac-(β2hAla)6-LysH+ to form native like helical structures in the gas phase. The design follows the known properties of the peptide Ac-Ala6-LysH+ that forms a α helix in isolation. We perform ion-mobility mass-spectrometry and vibrational spectroscopy in the gas phase, combined with state-of-the-art density-functional theory simulations of these molecular systems in order to characterize their structure. We can show that the straightforward exchange of alanine residues for the homologous β amino acids generates a system that is generally capable of adopting native like helices with backward oriented H-bonds. By pushing the limits of theory and experiments, we show that one cannot assign a single preferred structure type due to the densely populated energy landscape and present an interpretation of the data that suggests an equilibrium of three helical structures

Identification of Quantitative Trait Loci for Flowering Time in a Field-Grown \u3cem\u3eLolium Perene x Lolium Multiflorum\u3c/em\u3e Mapping Population

Perennial ryegrass (L. perenne) and annual, or Italian, ryegrass (L. multiflorum) are considered to be separate species by the seed trade, and are used and bred for distinct purposes. However, the two species are cross-fertile. Seed producers rely on the different flowering times of the two species to produce pure seed. Flowering times can overlap, leading to genetic mixing. Contamination of perennial ryegrass seed lots with annual types is an expensive problem for grass seed producers in western Oregon, USA