Improvement of in-gel digestion protocol for peptide mass fingerprinting by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

High-sensitivity, high-throughput analysis of proteins for proteomics studies is usually performed by polyacrylamide gel electrophoresis in combination with mass spectrometry. However, the quality of the data obtained depends on the in-gel digestion procedure employed. This work describes an improvement in the in-gel digestion efficiency for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) analysis. A dramatic improvement in the coverage of tryptic peptides was observed when n-octyl glucoside was added to the buffer. Whole cell extracted proteins from S. cerevisiae were separated by two-dimensional gel electrophoresis and stained with silver. Protein spots were identified using our improved in-gel digestion method and MALDI-TOFMS. In addition, the mass spectra obtained by using the matrix alpha-cyano-4-hydroxycinnamic acid (CHCA) were compared with those obtained using 2,5-dihydroxybenzoic acid (DHB). The DHB matrix usually gave more peaks, which led to higher sequence coverage and, consequently, to higher confidence in protein identification. This improved in-gel digestion protocol is simple and useful for protein identification by MALDI-TOFMS.     

Voltammetry at partially covered electrodes: Part III. Faradaic impedance measurements at model electrodes

Faradaic, impedances at model electrodes partially covered with a photoresist layer have been studied theoretically and experimentally. Equations for the faradaic impedance are derived based on the theoretical model and approach described in Part I of this series of papers. Experimental data for the hexacyanoferrate system at various model electrodes give excellent agreement, with theoretical predictions for the diffusion impedance behavior, and the applicability of the derived equations to the estimation of the degree of coverage and the size of the active regions is confirmed. Furthermore, the application of such model electrodes to the kinetic study of electrode reactions with high heterogeneous charge transfer rates is suggested.     

Electronic and ionic transports for negative charge carriers in smectic liquid crystalline photoconductor

We have investigated negative charge carrier transport in the smectic mesophases of the 2-phenylnaphthalene derivative, 6-(4'-octylphenyl)-2-dodecyloxynaphthalene (8-PNP-O12), using the time-of-flight (TOF) method. We revealed that the negative charge carrier transport in its smectic mesophases had two different mechanisms, i.e., electronic and ionic conductions: we observed two transits of the carriers in both the smectic A (SmA) and smectic B (SmB) phases and demonstrated their origins by dilution experiments with a hydrocarbon (n-dodecane); the fast transit was attributed to the electronic transport of electrons and the slow one to the ionic transport of negative ions. Furthermore, it was clarified that the ionic transport was caused by small amounts of chemical impurities ionized by trapping photogenerated electrons in 8-PNP-O12 in addition to photoinduced autoionization of the impurities. Furthermore, we determined the trapping lifetimes for electrons to be 140 and 24 mus for the SmA and SmB phases, respectively. The experimental results suggest the coexistence of two distinctive transport channels for these charge carriers in the smectic mesophases.     

Palladium(0)-catalyzed stereoselective cyclization of allenenes: divergent synthesis of pyrrolidines and 3-azabicyclo[3.1.0]hexanes from single allenenes

Two novel palladium(0)-catalyzed cyclizations of allenenes are described. Treatment of allenenes such as N-(1-alkyl-2,3-butadienyl)-N-allylsulfonamide with an aryl halide and K(2)CO(3) in the presence of a catalytic amount of Pd(PPh(3))(4) in dioxane affords 2,3-cis-pyrrolidines in a stereoselective manner. In sharp contrast, cyclization of the same allenenes using catalytic Pd(2)(dba)(3) x CHCl(3) in the presence of allyl methyl carbonate in CH(3)CN leads to stereoselective formation of a 3-azabicyclo[3.1.0]hexane framework in moderate yields.     

Radical cyclization by ipso substitution of the methoxy group: considerable effect of HMPA on samarium-mediated cyclization

Alkyl radicals generated by treatment of thiocarbamates of conformationally favorable 3-alkyl-3-arylpropan-1-ols with tris(trimethylsilyl)silane and AIBN efficiently undergo intramolecular ipso substitution of the methoxy group, yielding the corresponding cyclized products. In contrast, either conformationally favorable or flexible 1-arylalkan-3- or 4-ones easily cyclize into five- or six-membered condensed rings by treatment with SmI(2) via ketyl radical intermediates. The addition of HMPA as cosolvent dramatically changes the cyclization mode of the SmI(2)-induced reaction, and the para-cyclization products are exclusively formed. This "HMPA effect" can be rationalized by the strong chelating ability of HMPA with the samarium atom.     

Structural assignment of isomeric 2-aminopyridine-derivatized oligosaccharides using negative-ion MSn spectral matching

To investigate the possibility of structural assignment based on negative-ion MS2 spectral matching, three isomeric pairs of 2-aminopyridine (PA)-derivatized non-fucosylated, fucosylated, and sialylated oligosaccharides (complex type N-glycans) were analyzed using high-performance liquid chromatography/ion trap mass spectrometry (HPLC/ITMS) with a sonic-spray ionization (SSI) source. In the SSI negative-ion mode the deprotonated molecule [M-2H]2- becomes prominent. Negative-ion MS2 spectra derived from such ions contain many fragment types (B and Y, C and Z, A, and D) and therefore are more informative than the positive-ion MS2 spectra derived from [M+H+Na]2+ ions, which usually consist mainly of B and Y fragment ions. In particular the internal ions (D- and E-type ions) provided useful information about the alpha1-6 branching patterns and the bisecting GlcNAc residue. Spectral matching based on the correlation coefficients between negative-ion MS2 spectra was performed in a manner similar to the positive-ion MS2 spectral matching previously reported. It was demonstrated that negative-ion MS2 spectral matching is as useful and applicable to the structural assignment of relatively large non-fucosylated, fucosylated, and sialylated PA-oligosaccharide isomers as its positive-ion counterpart.     

The effect of fluorination: a crystallographic and computational study of mesogenic alkyl 4-[2-(perfluorooctyl)ethoxy]benzoates

The series of alkyl 4-[2-(perfluorooctyl)ethoxy]benzoates (F8-n) shows a systematic change of crystal structures depending on the length of the alkyl chain: separate packing of perfluorooctyl (Rf) and alkyl (Rh) chains from each other for shorter (n=2) and longer (n=11) members, alternate packing of Rf and Rh chains for middle (n=6,7) members, and an intermediate type of packing for n=4. Semiempirical MO calculations show slightly repulsive interactions between the Rf chains, and attractive ones between Rf and Rh chains and between Rh and the core of a molecular pair. It is concluded that fluorination determines the molecular shape of the crystal structures by making the chain rigid. It is confirmed that the interactions between Rf chains are small compared with those between other moieties and that they are forced to aggregate owing to the exclusion from other moieties. Thus, the effect is dependent on the geometries and intermolecular interactions of the other moieties.     

Laser-induced fluorescence of cyclohexadienyl (c-C6H7) radical in the gas phase

A laser-induced fluorescence spectrum was observed in the 500-560 nm region when a mixture of 1,4-cyclohexadiene and oxalyl chloride was photolyzed at 193 nm. The observed excitation spectrum was assigned to the A (2)A(2)<--X (2)B(1) transition of the cyclohexadienyl radical c-C6H7, produced by abstraction of a hydrogen atom from 1,4-cyclohexadiene by Cl atoms. The origin of the A<--X transition of c-C(6)H(7) was at 18 207 cm(-1). From measurements of the dispersed fluorescence spectra and ab initio calculations, the frequencies of several vibrational modes in both the ground and excited states of c-C(6)H(7) were determined: nu(5)(C-H in-plane bend)=1571, nu(8)(C-H in-plane bend)=1174, nu(10)(C-C-C in-plane bend)=981, nu(12)(C-C-C in-plane bend)=559, nu(16)(C-C-C out-of-plane bend)=375, and nu(33)(C-C-C in-plane bend)=600 cm(-1) for the ground state and nu(8)=1118, nu(10)=967, nu(12)=502, nu(16)=172, and nu(33)=536 cm(-1) for the excited states.     

Photochemical study of [3(3)](1,3,5)cyclophane and emission spectral properties of [3n]cyclophanes (n = 2-6)

Thephotochemical reaction of [3(3)](1,3,5)cyclophane 2, which is a photoprecursor for the formation of propella[3(3)]prismane 18, was studied using a sterilizing lamp (254 nm). Upon photolysis in dry and wet CH2Cl2 or MeOH in the presence of 2 mol/L aqueous HCl solution, the cyclophane 2 afforded novel cage compounds comprised of new skeletons, tetracyclo[6.3.1.0.(2,7)0(4,11)]dodeca-5,9-diene 43, hexacyclo[6.4.0.0.(2,6)0.(4,11)0.(5,10)0(9,12)]dodecane 44, and pentacyclo[6.4.0.0.(2,6)0.(4,11)0(5,10)]dodecane 45. All of these products were confirmed by the X-ray structural analyses. A possible mechanism for the formation of these photoproducts via the hexaprismane derivative 18 is proposed. The photophysical properties in the excited state of the [3n]cyclophanes ([3n]CP, n = 2-6) were investigated by measuring the emission spectra and determining the quantum yields and lifetimes of the fluorescence. All [3n]CPs show excimeric fluorescence without a monomeric one. The lifetime of the excimer fluorescence becomes gradually longer with the increasing number of the trimethylene bridges. The [3n]CPs also shows excimeric phosphorescence spectra without vibrational structures for n = 2, 4, and 5, while phosphorescence is absent for n = 3 and 6. With an increase in symmetry of the benzene skeleton in the [3(3)]- and [3(6)]CPs, the probability of the radiation (phosphorescence) process from the lowest triplet state may drastically decrease.