FPOP-LC-MS/MS Suggests Differences in Interaction Sites of Amphipols and Detergents with Outer Membrane Proteins

Amphipols are a class of novel surfactants that are capable of stabilizing the native state of membrane proteins. They have been shown to be highly effective, in some cases more so than detergent micelles, at maintaining the structural integrity of membrane proteins in solution, and have shown promise as vehicles for delivering native membrane proteins into the gas phase for structural interrogation. Here, we use fast photochemical oxidation of proteins (FPOP), which irreversibly labels the side chains of solvent-accessible residues with hydroxyl radicals generated by laser photolysis of hydrogen peroxide, to compare the solvent accessibility of the outer membrane protein OmpT when solubilized with the amphipol A8-35 or with n-dodecyl-β-maltoside (DDM) detergent micelles. Using quantitative mass spectrometry analyses, we show that fast photochemical oxidation reveals differences in the extent of solvent accessibility of residues between the A8-35 and DDM solubilized states, providing a rationale for the increased stability of membrane proteins solubilized with amphipol compared with detergent micelles, as a result of additional intermolecular contacts.

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Determination of eight polyphenols and pantothenic acid in extra‐virgin olive oil samples by a simple,fast, high‐throughput and sensitive ultra high performance liquid chromatography with tandem mass spectrometry method

A new ultra high performance liquid chromatography coupled with tandem mass spectrometry method for a fast and sensitive determination of eight polyphenols (hydroxytyrosol, catechin, epicatechin, epigallocatechin gallate, oleuropein, quercetin, rutin, tyrosol) and panthotenic acid in extra‐virgin olive oil was developed. The method does not require long sample pre‐treatment and presents the lowest limit of detection and limit of quantitation values present in literature. Inter‐ and intra‐day variability, linear dynamic range of the calibration curve, recovery and matrix effect were also determined and investigated. The method was applied to several oil samples of different type and origin. Given its accuracy, precision and rapidity, the method is characterized by an interestingly high throughput, reliability, and sensitivity.     

Biocompatible Infinite‐Coordination‐Polymer Nanoparticle–Nucleic‐Acid Conjugates for Antisense Gene Regulation

Herein, we report the synthesis of DNA‐functionalized infinite‐coordination‐polymer (ICP) nanoparticles as biocompatible gene‐regulation agents. ICP nanoparticles were synthesized from ferric nitrate and a ditopic 3‐hydroxy‐4‐pyridinone (HOPO) ligand bearing a pendant azide. Addition of Fe^III to a solution of the ligand produced nanoparticles, which were colloidally unstable in the presence of salts. Conjugation of DNA to the Fe^III–HOPO ICP particles by copper‐free click chemistry afforded colloidally stable nucleic‐acid nanoconstructs. The DNA–ICP particles, when cross‐linked through sequence‐specific hybridization, exhibited narrow, highly cooperative melting transitions consistent with dense DNA surface loading. The ability of the DNA–ICP particles to enter cells and alter protein expression was also evaluated. Our results indicate that these novel particles carry nucleic acids into mammalian cells without the need for transfection agents and are capable of efficient gene knockdown.     

Correlation functions of the one-dimensional attractive Bose gas

The zero-temperature correlation functions of the one-dimensional attractive Bose gas with a delta-function interaction are calculated analytically for any value of the interaction parameter and number of particles, directly from the integrability of the model. We point out a number of interesting features, including zero recoil energy for a large number of particles, analogous to the M?ssbauer effect.     

A General Treatment to Study Molecular Complexes Stabilized by Hydrogen‐, Halogen‐, and Carbon‐Bond Networks: Experiment and Theory of (CH2F2)n⋅⋅⋅(H2O)m

Rotational spectra of several difluoromethane–water adducts have been observed using two broadband chirped‐pulse Fourier‐transform microwave (CP‐FTMW) spectrometers. The experimental structures of (CH₂F₂)???(H₂O)₂, (CH₂F₂)₂???(H₂O), (CH₂F₂)???(H₂O)₃, and (CH₂F₂)₂???(H₂O)₂ were unambiguously identified with the aid of 18 isotopic substituted species. A subtle competition between hydrogen, halogen, and carbon bonds is observed and a detailed analysis was performed on the complex network of non‐covalent interactions which stabilize each cluster. The study shows that the combination of stabilizing contact networks is able to reinforce the interaction strength through a cooperative effect, which can lead to large stable oligomers.     

Proteomic characterization of donkey milk “caseome”

At present, compared with bovine milk, the characterization of donkey milk caseins is at a relatively early stage progress, and only limited data are related to its genetic polymorphism. In this work, the heterogeneity of donkey caseome was investigated using a proteomic approach, based on one- (PAGE, UTLIEF) and two-dimensional (PAGE → UTLIEF) electrophoresis, stained with either Coomassie Brilliant Blue or specific polyclonal antibodies, and structural MS analysis. These combined methodologies allowed the contemporary identification of donkey α_s1, α_s2, β and κ-CN with their related heterogeneity due to phosphorylation (α_s1, α_s2 and β-CN), glycosylation (κ-CN) and incorrect splicing of RNA in mRNA (deleted forms of α_s1-CN and β-CN). The results achieved showed 11 components for κ-CN, six phosphorylated components for β and α_s1-CN and three main phosphorylated components for α_s2-CN, each accounting for 10, 11 and 12 P/mole. At this regard, for the first time, the primary structure of the expressed protein corresponding to the only available donkey α_s2-CN cDNA sequence was determined. Furthermore β-CN was found in homozygous and heterozygous state for the occurrence of a genetic β-CN variant having a MW value 28 mass units higher than the common β-CN phenotype.     

Ultrafast continuum mid-infrared spectroscopy: probing the entire vibrational spectrum in a single laser shot with femtosecond time resolution

Until now, ultrafast IR spectroscopy has been limited by the bandwidth of optical parametric amplifiers, typically 100-400 cm(-1). Here we present the first example of transient IR spectroscopy using a continuum laser source to probe the entire mid-IR region with ultrafast time resolution. The continuum source is based on focusing the fundamental, second harmonic, and third harmonic of 1 mJ, 25 fs, 800 nm pulses in air, generating ～150 fs continuum mid-IR pulses that span the frequency range of <400 to >5000 cm(-1) or, conversely, <2 to >25 μm. We characterize the spectral and temporal properties of dicarbonylacetonato rhodium(I) in hexane. We further demonstrate the versatility of the method by measuring the very fast and broad (>1500 cm(-1)) spectral changes following IR excitation associated with the 7-azaindole-acetic acid heterodimer in carbon tetrachloride.     

A Negative Ion Mass Spectrometry Approach to Identify Cross-Linked Peptides Utilizing Characteristic Disulfide Fragmentations

Chemical cross-linking combined with mass spectrometry (MS) is an analytical tool used to elucidate the topologies of proteins and protein complexes. However, identification of the low abundance cross-linked peptides and modification sites amongst a large quantity of proteolytic fragments remains challenging. In this work, we present a strategy to identify cross-linked peptides by negative ion MS for the first time. This approach is based around the facile cleavages of disulfide bonds in the negative mode, and allows identification of cross-linked products based on their characteristic fragmentations. MS(3) analysis of the cross-linked peptides allows for their sequencing and identification, with residue specific location of cross-linking sites. We demonstrate the applicability of the commercially available cystine based cross-linking reagent dithiobis(succinimidyl) propionate (DSP) and identify cross-linked peptides from ubiquitin. In each instance, the characteristic fragmentation behavior of the cross-linked species is described. The data presented here indicate that this negative ion approach may be a useful tool to characterize the structures of proteins and protein complexes, and provides the basis for the development of high throughput negative ion MS chemical cross-linking strategies.     

Time dependence of correlation functions following a quantum quench

We show that the time dependence of correlation functions in an extended quantum system in d dimensions, which is prepared in the ground state of some Hamiltonian and then evolves without dissipation according to some other Hamiltonian, may be extracted using methods of boundary critical phenomena in d + 1 dimensions. For d = 1 particularly powerful results are available using conformal field theory. These are checked against those available from solvable models. They may be explained in terms of a picture, valid more generally, whereby quasiparticles, entangled over regions of the order of the correlation length in the initial state, then propagate classically through the system.