d(GCGAAGC) is the smallest oligonucleotide with a well-defined hairpin structure in solution. We report a study of multiply protonated d(GCGAAGC) and its sequence-scrambled isomers, d(CGAAGCG), d(GCGAACG), and d(CGGAAGC), that were produced by electrospray ionization with the goal of investigating their gas-phase structures and dissociations. Cyclic ion mobility measurements revealed that dications of d(GCGAAGC) as well as the scrambled-sequence ions were mixtures of protomers and/or conformers that had collision cross sections (CCS) within a 439–481 Å2 range. Multiple ion conformers were obtained by electrospray under native conditions as well as from aqueous methanol. Arrival time distribution profiles were characteristic of individual isomeric heptanucleotides. Extensive Born–Oppenheimer molecular dynamics (BOMD) and density functional theory (DFT) calculations of d(GCGAAGC)2+ isomers indicated that hairpin structures were high-energy isomers of more compact distorted conformers. Protonation caused a break up of the C2···G6 pair that was associated with the formation of strong hydrogen bonds in zwitterionic phosphate anion-nucleobase cation motifs that predominated in low energy ions. Multiple components were also obtained for d(GCGAAGC)3+ trications under native and denaturing electrospray conditions. The calculated trication structures showed disruption of the G···C pairs in low energy zwitterions. A hairpin trication was calculated to be a high energy isomer. d(GCGAAGC)4+ tetracations were produced and separated by c-IMS as two major isomers. All low energy d(GCGAAGC)4+ ions obtained by DFT geometry optimizations were zwitterions in which all five purine bases were protonated, and the ion charge was balanced by a phosphate anion. Tetracations of the scrambled sequences were each formed as one dominant isomer. The CCS calculated with the MobCal-MPI method were found to closely match experimental values. Collision-induced dissociation (CID) spectra of multiply charged heptanucleotides showed nucleobase loss and backbone cleavages occurring chiefly at the terminal nucleosides. Electron-transfer-CID tandem mass spectra were used to investigate dissociations of different charge and spin states of charge-reduced heptanucleotide cation radicals.
Abstract The octanol‐water partition coefficients of seven anthocyanins (in neutral form, pH = 7.0) were measured by means of MEKC. A new iterative method for the estimation of migration time of electroosmotic flow (EOF) and micelles in MEKC is presented. This calculation is based on Gauss‐Newton linearization of the dependence between migration indices and migration times of a set of suitable standards and the application of an advanced statistical evaluation procedure. The values of partition coefficients obtained with the aid of an iterative process are compared with values obtained on use of EOF and micelle markers.
Applicability of mass spectrometry is related to the progress in instrumentation including ion sources. Ionization techniques in MS often need sample preparation, which increases the analysis time. From this point of view, DESI (desorption electrospray ionization) seems to be an interesting alternative. Electrospraying of a sample with a liquid leads to the production of analyte ions desorbed from the surface-deposited sample. A new modification of DESI, where electrospraying is provided by nano-ESI, and a new application in chiral analysis have been developed. Chiral analysis employs the Cooks kinetic method. Chiral selector is sprayed by nano-ESI onto the surface with a deposited chiral analyte. Trimeric clusters are formed in the bulk of the ion source. Their fragmentation allows chiral discrimination of enantiomers. The power of the DESI-based mass spectrometric tool is demonstrated on chiral analysis of ephedrine in commercial pharmaceutical Mucoseptonex® E. The analysis is performed without any sample preparation. That is why the analysis is simple and cost-effective. Calibration for ephedrine was performed with D-enantiomer and the racemate (correlation coefficient R2 = 0.996) was obtained.
Nitrile imines produced by photodissociation of 2,5-diaryltetrazoles undergo cross-linking reactions with amide groups in peptide-tetrazole (tet-peptide) conjugates and a tet-peptide-dinucleotide complex. Tetrazole photodissociation in gas-phase ions is efficient, achieving ca. 50% conversion with 2 laser pulses at 250 nm. The formation of cross-links was detected by CID-MS3 that showed structure-significant dissociations by loss of side-chain groups and internal peptide segments. The structure and composition of cross-linking products were established by a combination of UV–vis action spectroscopy and cyclic ion mobility mass spectrometry (c-IMS). The experimental absorption bands were found to match the bands calculated for vibronic absorption spectra of nitrile imines and cross-linked hydrazone isomers. The calculated collision cross sections (CCSth) for these ions were related to the matching experimental CCSexp from multipass c-IMS measurements. Loss of N2 from tet-peptide conjugates was calculated to be a mildly endothermic reaction with ΔH0 = 80 kJ mol–1 in the gas phase. The excess energy in the photolytically formed nitrile imine is thought to drive endothermic proton transfer, followed by exothermic cyclization to a sterically accessible peptide amide group. The exothermic nitrile imine reaction with peptide amides is promoted by proton transfer and may involve an initial [3 + 2] cycloaddition followed by cleavage of the oxadiazole intermediate. Nucleophilic groups, such as cysteine thiol, did not compete with the amide cyclization. Nitrile imine cross-linking to 2′-deoxycytidylguanosine was found to be >80% efficient and highly specific in targeting guanine. The further potential for exploring nitrile-imine cross-linking for biomolecular structure analysis is discussed.
Crude extract of Aspergillus niger AKU 3302 mycelia incubated with methylamine showed a single amine oxidase activity band in a developed polyacrylamide gel that weakly cross-reacted with the antibody against a copper/topa quinone-containing amine oxidase (AO-II) from the same strain induced by n-butylamine. Since the organism cannot grow on methylamine and the already known quinoprotein amine oxidases of the organism cannot catalyze oxidation of methylamine, the organism was forced to produce another enzyme that could oxidize methylamine when the mycelia were incubated with methylamine. The enzyme was separated and purified from the already known two quinoprotein amine oxidases formed in the same mycelia. The purified enzyme showed a sharp symmetric sedimentation peak in analytical ultracentrifugation showing S20,w0 of 6.5s. The molecular mass of 133 kDa estimated by gel chromatography and 66.6 kDa found by SDS-PAGE confirmed the dimeric structure of the enzyme. The purified enzyme was pink in color with an absorption maximum at 494 nm. The enzyme readily oxidized methylamine, n-hexylamine, and n-butylamine, but not benzylamine, histamine, or tyramine, favorite substrates for the already known two quinoprotein amine oxidases. Inactivation by carbonyl reagents and copper chelators suggested the presence of a copper/topa quinone cofactor. Spectrophotometric titration by p-nitrophenylhydrazine showed one reactive carbonyl group per subunit and redox-cyclic quinone staining confirmed the presence of a quinone cofactor. pH-dependent shift of the absorption spectrum of the enzyme-p-nitrophenylhydrazone (469 nm at neutral to 577 nm at alkaline pH) supported the identity of the cofactor with topaquinone. Nothern blot analysis indicated that the methylamine oxidase encoding gene is largely different from the already known amine oxidase in the organism.
