Protein identification by accurate mass matrix-assisted laser desorption/ionization imaging of tryptic peptides

The spatial distribution of proteins in tissue sections can be used to identify potential markers for pathological processes. Tissue sections are often subjected to enzymatic digestion before matrix‐assisted laser desorption/ionization (MALDI) imaging. This study is targeted at improving the on‐tissue identification of tryptic peptides by accurate mass measurements and complementary off‐line liquid chromatography coupled to electrospray ionization tandem mass spectrometry (LC/ESI‐MS/MS) analysis. Two adjacent mouse brain sections were analyzed in parallel. The first section was spotted with trypsin and analyzed by MALDI imaging. Direct on‐tissue MS/MS experiments of this section resulted in the identification of 14 peptides (originating from 4 proteins). The second tissue section was homogenized, fractionated by ultracentrifugation and digested with trypsin prior to LC/ESI‐MS/MS analysis. The number of identified peptides was increased to 153 (corresponding to 106 proteins) by matching imaged mass peaks to peptides which were identified in these LC/ESI‐MS/MS experiments. All results (including MALDI imaging data) were based on accurate mass measurements (RMS <2 ppm) and allow a confident identification of tryptic peptides. Measurements based on lower accuracy would have led to ambiguous or misleading results. MS images of identified peptides were generated with a bin width (mass range used for image generation) of Δm/z = 0.01. The application of accurate mass measurements and additional LC/MS measurements increased both the quality and the number of peptide identifications. The advantages of this approach for the analysis of biological tissue sections are demonstrated and discussed in detail. Results indicate that accurate mass measurements are needed for confident identification and specific image generation of tryptic peptides in tissue sections. Copyright © 2011 John Wiley & Sons, Ltd.     

S K-edge XAS and DFT calculations on SAM dependent pyruvate formate-lyase activating enzyme: nature of interaction between the Fe4S4 cluster and SAM and its role in reactivity

S K-edge X-ray absorption spectroscopy on the resting oxidized and the S-adenosyl-l-methionine (SAM) bound forms of pyruvate formate-lyase activating enzyme are reported. The data show an increase in pre-edge intensity, which is due to additional contributions from sulfide and thiolate of the Fe(4)S(4) cluster into the C-S σ* orbital. This experimentally demonstrates that there is a backbonding interaction between the Fe(4)S(4) cluster and C-S σ* orbitals of SAM in this inner sphere complex. DFT calculations that reproduce the data indicate that this backbonding is enhanced in the reduced form and that this configurational interaction between the donor and acceptor orbitals facilitates the electron transfer from the cluster to the SAM, which otherwise has a large outer sphere electron transfer barrier. The energy of the reductive cleavage of the C-S bond is sensitive to the dielectric of the protein in the immediate vicinity of the site as a high dielectric stabilizes the more charge separated reactant increasing the reaction barrier. This may provide a mechanism for generation of the 5'-deoxyadenosyl radical upon substrate binding.     

Alpha-particle reactions

Recent work using the concept of alpha-clustering in studies of nuclear structure and nuclear reactions is reviewed. The high symmetry and binding energy of the alpha-particle makes it likely that nucleons inside the nucleus can condense into alpha particles and live long enough to affect many properties of nuclei and also the cross-sections of nuclear reactions, particularly those with alpha-particles as the projectile or ejectile. The alpha-particles inside the nucleus may escape, and the resulting alpha decay is now quite well understood. An incident projectile may collide with a transient alpha-particle in the nucleus and knock it out, thus providing information on the degree of clustering. This model enables the direct part of the cross-sections of (n, α) reactions to be calculated. The alpha-particle mean field unifies many of the characteristics of alpha-particle structure and alpha-particle scattering. Many properties of light nuclei may be simply explained using the concept of alpha-clustering.     

A network location-allocation model trading off flow capturing andp-median objectives

The flow capturing and thep-median location—allocation models deal quite differently with demand for service in a network. Thep-median model assumes that demand is expressed at nodes and locates facilities to minimize the total distance between such demand nodes and the nearest facility. The flow-capturing model assumes that demand is expressed on links and locates facilities to maximize the one-time exposure of such traffic to facilities. Demand in a network is often of both types: it is expressed by passing flows and by consumers centred in residential areas, aggregated as nodes. We here present a hybrid model with the dual objective of serving both types of demand. We use this model to examine the tradeoff between serving the two types of demand in a small test network using synthetic demand data. A major result is the counter-intuitive finding that thep-median model is more susceptible to impairment by the flow capturing objective than is the flow capturing model to thep-median objective. The results encourage us to apply the model to a real-world network using actual traffic data.     

Multi-Service Facility Location Models

The multi-service facility (MSF) concept proposes the co-location of a range of human services under a single roof. Thus, for example, services for preschoolers, teens, and seniors might be co-located within a joint facility. MSFs are a response to economies of scale; co-location reduces the monetary costs of providing a variety of services. Yet, the spatial distributions of different societal groups are different within a city – an MSF system will not provide optimum geographical accessibility to individual groups. We introduce two p-median based location–allocation models that trade off the fixed costs of providing services and opening facilities with the travel costs of three societal groups. We observe that some mixes of single- and multi-service facilities can provide efficient service systems without unduly compromising the accessibility needs of individual groups.     

Modification of guanine residues in PNA-synthesis by PyBOP

The phosphonium-type coupling reagent PyBOP, when applied to the synthesis of peptide nucleic acid (PNA) oligomers, was found to form O⁴-phosphonium compounds of the nucleobase guanine which can be converted into C⁴-modified guanine-derived PNAs by nucleophiles.     

Multistep processes in nuclear reactions

Recent applications of the theory of Feshbach, Kerman and Koonin to analyse multistep processes in nuclear reactions are described, and illustrated by detailed comparisons with a range of experimental data for neutron and proton reactions. The techniques used to distinguish between the multistep direct, multistep compound, compound and collective contributions to the cross-sections are described, and their effectiveness assessed. Particular attention is devoted to recent analyses that take into account the transitions from the multistep direct to the multistep compound chain and also to those that evaluate the collective contributions to the continuum region. The variation of the effective nucleonnucleon interaction with incident energy and target nucleus is studied, and the possibility of a parameter-free calculation is discussed. The extensions of the theory to include multiparticle emission from the direct chain and also to studies of alpha-particle emission are described.     

Experimental test of the non-thermal theory of laser annealing using silicon on sapphire

The non-thermal, pulsed plasma model of laser annealing was experimentally tested by comparing the effects of 30 ns and 1 μs laser pulses on silicon on sapphire films, impinging from both the front and rear surfaces. The energy density needed to anneal the films and the qualitative behavior of thermal shock damages are in accordance with the simple heating model and inconsistent with the non-thermal model.