Laser-Induced Hydrogen Radical Removal in UV MALDI-MS Allows for the Differentiation of Flavonoid Monoglycoside Isomers

Negative-ion matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectra and tandem mass spectra of flavonoid mono-O-glycosides showed the irregular signals that were 1 and/or 2 Da smaller than the parent deprotonated molecules ([M – H]^–) and the sugar-unit lost fragment ions ([M – Sugar – H]^–). The 1 and/or 2 Da mass shifts are generated with the removing of a neutral hydrogen radical (H*), and/or with the homolytic cleavage of the glycosidic bond, such as [M – H* – H]^–, [M – Sugar – H* – H]^–, and [M – Sugar – 2H* – H]^–. It was revealed that the hydrogen radical removes from the phenolic hydroxy groups on the flavonoids, not from the sugar moiety, because the flavonoid backbones themselves absorb the laser. The glycosyl positions depend on the extent of the hydrogen radical removals and that of the homolytic cleavage of the glycosidic bonds. Flavonoid mono-glycoside isomers were distinguished according to their TOF MS and tandem mass spectra.

Mobile Proton Triggered Radical Fragmentation of Nitroarginine Containing Peptides

Protonated nitroarginine, [R^NO2 + H]⁺, which contains the nitroguanidine ‘explosophore,’ undergoes homolytic N – N nitro-imine bond cleavage to expel NO₂ ^? and form a radical cation of arginine in high yield (100 % relative abundance) upon low-energy collision-induced dissociation (CID). Other ionization states of nitroarginine, including [R^NO2 - H]^–, and a fixed-charge derivative of nitroarginine do not expel NO₂ ^? (<1 %), but instead dissociate via heterolytic bond cleavage with abundant losses of small molecules (N₂O and H₂N₂O₂) from the nitroguanidine group. The effects of proton mobility on the CID reactions of nitroarginine containing peptides was investigated for peptide derivatives of leucine enkephalin, including XYGGFLR^NO2, X = D, G, K, and R, by examining the different protonation states: [M – H]^–; [M + H]⁺; and [M + 2H]²⁺. For [M + H]⁺ containing the less basic N-terminal residues (X = D, G) and all [M + 2H]²⁺, mobile proton fragmentation reactions that result in peptide sequence ions dominate. In contrast, for peptides containing the basic N-terminal residues (R and K), the CID spectra of both the [M – H]^– and [M + H]⁺ are dominated by the losses of small even-electron neutrals from the nitroarginine side-chain. The fraction of nitroguanidine directed fragmentation of the nitroarginine side chain that results in bond homolysis to form [XYGGFLR]^+? by expulsion of NO₂ ^? increases by more than 10 times as the protonation state changes from [M – H]^– (<10 %) to [M + 2H]²⁺ (ca. 90 %) and by about four times as the acidity of the [M + H]⁺ N-terminal residue increases from R (19.0 %) to D (76.5 %). These results indicate that protonated peptides containing nitroarginine can undergo non-canonical mobile proton triggered radical fragmentation.

Mechanistic Investigation of Phosphate Ester Bond Cleavages of Glycylphosphoserinyltryptophan Radical Cations under Low-Energy Collision-Induced Dissociation

Under the conditions of low-energy collision-induced dissociation (CID), the canonical glycylphosphoserinyltryptophan radical cation having its radical located on the side chain of the tryptophan residue ([G _p SW]^?+) fragments differently from its tautomer with the radical initially generated on the α-carbon atom of the glycine residue ([G^? _p SW]⁺). The dissociation of [G^? _p SW]⁺ is dominated by the neutral loss of H₃PO₄ (98 Da), with backbone cleavage forming the [b₂ – H]^?+/y₁ ⁺ pair as the minor products. In contrast, for [G _p SW]^?+, competitive cleavages along the peptide backbone, such as the formation of [G _p SW – CO₂]^?+ and the [c₂?+?2H]⁺/[z₁ – H]^?+ pair, significantly suppress the loss of neutral H₃PO₄. In this study, we used density functional theory (DFT) to examine the mechanisms for the tautomerizations of [G^? _p SW]⁺ and [G _p SW]^?+ and their dissociation pathways. Our results suggest that the dissociation reactions of these two peptide radical cations are more efficient than their tautomerizations, as supported by Rice–Ramsperger–Kassel–Marcus (RRKM) modeling. We also propose that the loss of H₃PO₄ from both of these two radical cationic tautomers is preferentially charge-driven, similar to the analogous dissociations of even-electron protonated peptides. The distonic radical cationic character of [G^? _p SW]⁺ results in its charge being more mobile, thereby favoring charge-driven loss of H₃PO₄; in contrast, radical-driven pathways are more competitive during the CID of [G _p SW]^?+.

Structural Distinction of Diacyl-, Alkylacyl,and Alk-1-Enylacyl Glycerophosphocholines as [M – 15]– Ions by Multiple-Stage Linear Ion-Trap Mass Spectrometry with Electrospray Ionization

We describe a linear ion-trap (LIT) multiple-stage (MSⁿ) mass spectrometric approach towards differentiation of alkylacyl, alk-1-enylacyl- and diacyl-glycerophoscholines (PCs) as the [M – 15]^– ions desorbed by electrospray ionization (ESI) in the negative-ion mode. The MS⁴ mass spectra of the [M – 15 – R₂′CH = CO]^– ions originated from the three PC subfamilies are readily distinguishable, resulting in unambiguous distinction of the lipid classes. This method is applied to two alkyl ether rich PC mixtures isolated from murine bone marrow neutrophils and kidney, respectively, to explore its utility in the characterization of complex PC mixture of biological origin, resulting in the realization of the detailed structures of the PC species, including various classes and many minor isobaric isomers.

Electron Capture Dissociation Studies of the Fragmentation Patterns of Doubly Protonated and Mixed Protonated-Sodiated Peptoids

The fragmentation patterns of a group of doubly protonated ([P + 2H]²⁺) and mixed protonated-sodiated ([P + H + Na]²⁺) peptide-mimicking oligomers, known as peptoids, have been studied using electron capturing dissociation (ECD) tandem mass spectrometry techniques. For all the peptoids studied, the primary backbone fragmentation occurred at the N-C_α bonds. The N-terminal fragment ions, the C-ions (protonated) and the C′-ions (sodiated) were observed universally for all the peptoids regardless of the types of charge carrier. The C-terminal ions varied depending on the type of charge carrier. The doubly protonated peptoids with at least one basic residue located at a position other than the N-terminus fragmented by producing the Z^?-series of ions. In addition, most doubly protonated peptoids also produced the Y-series of ions with notable abundances. The mixed protonated-sodiated peptoids fragmented by yielding the Z^?′-series of ions in addition to the C′-series. Chelation between the sodium cation and the amide groups of the peptoid chain might be an important factor that could stabilize both the N-terminal and the C-terminal fragment ions. Regardless of the types of the charge carrier, one notable fragmentation for all the peptoids was the elimination of a benzylic radical from the odd-electron positive ions of the protonated peptoids ([P + 2H]^?+) and the sodiated peptoids ([P + H + Na]^?+). The study showed potential utility of using the ECD technique for sequencing of peptoid libraries generated by combinatorial chemistry.

Why do the Abundances of Ions Generated by MALDI Look Thermally Determined?

In a previous study (J. Mass Spectrom. 48, 299–305, 2013), we observed that the abundance of each ion in a matrix-assisted laser desorption ionization (MALDI) spectrum looked thermally determined. To find out the explanation for the phenomenon, we estimated the ionization efficiency and the reaction quotient (Q_A) for the autoprotolysis of matrix, M + M → [M + H]⁺ + [M ? H]^?, from the temperature-controlled laser desorption ionization spectra of α-cyano-4-hydroxycinnamic acid (CHCA) and 2,5-dihydroxybenzoic acid (DHB). We also evaluated the equilibrium constants (K_A) for the autoprotolysis at various temperatures by quantum chemical calculation. Primary ion formation via various thermal models followed by autoprotolysis-recombination was compatible with the observations. The upper limit of the effective temperature of the plume where autoprotolysis-recombination occurs was estimated by equating Q_A with the calculated equilibrium constant.

Solvent extraction of europium trifluoromethanesulfonate into nitrobenzene by using three electroneutral receptors

From extraction experiments and $ \gamma $ -activity measurements, the extraction constants corresponding to the general equilibrium Eu³⁺(aq) + 3 A^?(aq) + L(nb) $ \Leftrightarrow $ EuL³⁺(nb) + 3A^?(nb) taking place in the two-phase water–nitrobenzene system ( $ {\text{A}}^{ - } = {\text{CF}}_{ 3} {\text{SO}}_{3}^{ - } $ ; L = electroneutral receptors denoted by 1, 2, and 3 – see Scheme 1; aq = aqueous phase, nb = nitrobenzene phase) were evaluated. Further, the stability constants of the EuL³⁺ complexes in nitrobenzene saturated with water were calculated; they were found to increase in the series of 3 < 2 < 1.

Effect of mobile phase on resolution of the isomers and homologues of tocopherols on a triacontyl stationary phase

Reversed-phase liquid chromatographic (RPLC) separation of isomers and homologues of similar polarity is challenging. Tocopherol isomers and homologues are one such example. α, β, γ, and δ-tocopherols have been successfully separated by RPLC on triacontyl (C₃₀) stationary phase. System suitability was tested by using four mobile phases, and observed chromatographic separations of β and γ-tocopherols were compared. Comparison indicated that methanol–tert-butyl methyl ether (TBME) 95:5 (v/v) at a flow rate of 0.75 mL min^?1 was the best mobile phase. Detection systems were also evaluated on the basis of limit of quantification; it was concluded that fluorescence detection was best. The method was validated by analysis of two homologues and two isomers of tocopherol in sesame, maize, and soybean samples. MS coupled with an ESI interface in negative-ion mode [M ? H]^? was used for identification of individual components. It was concluded that addition of TBME to methanol was required to enhance the separation of β and γ-tocopherols, although methanol alone provided similar results. The applicability of the method to cereal, pulse, and oilseed samples was confirmed. The reproducibility of the procedure was good, with relative standard deviations in the range 1.7–3.9 %. Recovery of tocopherols added to sesame samples ranged from 91 to 99 %.

Quantification of Competing H3PO4 Versus HPO3 + H2O Neutral Losses from Regioselective 18O-Labeled Phosphopeptides

Abundant neutral losses of 98 Da are often observed upon ion trap CID-MS/MS of protonated phosphopeptide ions. Two competing fragmentation pathways are involved in this process, namely, the direct loss of H₃PO₄ from the phosphorylated residue and the combined losses of HPO₃ and H₂O from the phosphorylation site and from an additional site within the peptide, respectively. These competing pathways produce product ions with different structures but the same m/z values, potentially limiting the utility of CID-MS³ for phosphorylation site localization. To quantify the relative contributions of these pathways and to determine the conditions under which each pathway predominates, we have examined the ion trap CID-MS/MS fragmentation of a series of regioselective ¹⁸O-phosphate ester labeled phosphopeptides prepared using novel solution-phase amino acid synthesis and solid-phase peptide synthesis methodologies. By comparing the intensity of the –100 Da (–H₃PO₃ ¹⁸O) versus –98 Da (–[HPO₃ + H₂O]) neutral loss product ions formed upon MS/MS, quantification of the two pathways was achieved. Factors that affect the extent of formation of the competing neutral losses were investigated, with the combined loss pathway predominantly occurring under conditions of limited proton mobility, and with increased combined losses observed for phosphothreonine compared with phosphoserine-containing peptides. The combined loss pathway was found to be less dominant under ion activation conditions associated with HCD-MS/MS. Finally, the contribution of carboxylic acid functional groups and backbone amide bonds to the water loss in the combined loss fragmentation pathway was determined via methyl esterification and by examination of a phosphopeptide lacking side-chain hydroxyl groups.

Reagent Cluster Anions for Multiple Gas-Phase Covalent Modifications of Peptide and Protein Cations

Multiple gas phase ion/ion covalent modifications of peptide and protein ions are demonstrated using cluster-type reagent anions of N-hydroxysulfosuccinimide acetate (sulfo-NHS acetate) and 2-formyl-benzenesulfonic acid (FBMSA). These reagents are used to selectively modify unprotonated primary amine functionalities of peptides and proteins. Multiple reactive reagent molecules can be present in a single cluster ion, which allows for multiple covalent modifications to be achieved in a single ion/ion encounter and at the ‘cost’ of only a single analyte charge. Multiple derivatizations are demonstrated when the number of available reactive sites on the analyte cation exceeds the number of reagent molecules in the anionic cluster (e.g., data shown here for reactions between the polypeptide [K₁₀ + 3H]³⁺ and the reagent cluster [5R_5Na – Na]^–). This type of gas-phase ion chemistry is also applicable to whole protein ions. Here, ubiquitin was successfully modified using an FBMSA cluster anion which, upon collisional activation, produced fragment ions with various numbers of modifications. Data for the pentamer cluster are included as illustrative of the results obtained for the clusters comprised of two to six reagent molecules.

Selective isolation of hemoglobin by use of imidazolium-modified polystyrene as extractant

Ionic liquids have attracted much attention in the analysis of a variety of species. The functional groups in ionic liquids can result in highly efficient separation and enrichment and, because of their typical lack of volatility, they are environmentally benign. We grafted imidazole cations onto the surface of chloromethyl polystyrene, denoted PS-CH₂-[MIM]⁺Cl^?, and this modified polymer was used to selectively extract the protein hemoglobin (Hb). The prepared extractant PS-CH₂-[MIM]⁺Cl^?, containing 2 mmol immobilized imidazole groups per gram polymer, was characterized by FT-IR, surface charge analysis, and elemental analysis. The adsorption efficiency was 91 %. The adsorption capacity of the PS-CH₂-[MIM]⁺Cl^? for Hb was 23.6 μg mg^?1, and 80 % of the retained Hb could be readily recovered by use of 0.5 % (m/v) aqueous sodium dodecyl sulfate (SDS) solution as eluate. The activity of the eluted Hb was approximately 90 %. The prepared imidazole-containing solid phase polymer was used for direct adsorption of Hb without use of any other solid matrix as support of the ionic liquid. The material was used in practice to isolate Hb from human whole blood.

O−• chemical ionization of carbonyl compounds

The negative ion chemical ionization mass spectra of twentyeight C₄ to C₇ carbonyl compounds were recorded using the oxide radical anion O^?? as reagent ion. As noted earlier, the reactions occurring include H⁺ abstraction, H ₂ ^+? abstraction, H? atom displacement, and alkyl radical displacement. In addition, the [M?2H]^? ions fragment further by alkyl radical elimination. The relative importance of these reactions depends strongly on molecular structure, with the result that isomer distinction frequently is possible. Where this is not possible, as for isomeric aldehydes, the collisional charge inversion mass spectra of common product ions provides isomer distinction. The H ₂ ^+? abstraction reaction is shown to involve abstraction not only of two hydrogens from the same α-carbon but also, in part, abstraction of one hydrogen from each α-carbon.     

Exploiting thermodynamic data to optimize the enantioseparation of underivatized amino acids in ligand exchange capillary electrophoresis

Stereoselective amino acid analysis has increasingly moved into the scope of interest of the scientific community. In this work, we report a study on the chiral separation of underivatized d,l-His by ligand exchange capillary electrophoresis (LECE), utilizing accurate ex ante calculations. This has been obtained by the addition to the background electrolytes (BGE) of NaClO₄ which renders the separations “all in solution processes”, allowing to accurately calculate in advance the concentrations of the species present in solution and to optimize the system performances. To this aim, the formation of ternary complexes of Cu²⁺ ion and l-lysine (l-Lys) or l-ornithine (l-Orn) with l- and d-histidine (His), and histamine (Hm) have been studied by potentiometry and calorimetry at 25 °C and with 0.1 mol dm^?3 (KNO₃) in aqueous solution. The ternary species [Cu(L)(l-His)H]⁺ and [Cu(L)(d-His)H]⁺ (where L?=?l-Lys or l-Orn) show a slight but still detectable stereoselectivity, and the determination of ΔH° and ΔS° values allowed the understanding of the factors which determine this phenomenon. The stereoselectivity showed by the protonated ternary species has been exploited to chirally separate d,l-His in LECE, by using the binary complexes of copper(II) with l-Lys or l-Orn as background electrolytes added with the appropriate amounts of NaClO₄.

A lanthanide nanoparticle-based luminescent probe for folic acid

We report on a novel luminescent method for the detection of folic acid (FA), a member of the vitamin B family. Y₂O₃ nanoparticles were doped with europium(III) ions and surface-modified with captopril. Their fluorescence is quenched by FA, and intensity is a function of folic acid concentration in the 0.1 – 40 μM concentration range. The detection limit is 83 nM of FA at pH 7 and room temperature.

Preparation of MIP grafts for quercetin by tandem aryl diazonium surface chemistry and photopolymerization

The food antioxidant quercetin was used as a template in an ultrathin molecularly imprinted polymer (MIP) film prepared by photopolymerization. Indium tin oxide (ITO) plates were electrografted with aryl layers via a diazonium salt precursor bearing two terminal hydroxyethyl groups. The latter act as hydrogen donors for the photosensitizer isopropylthioxanthone and enabled the preparation of MIP grafts through radical photopolymerization of methacrylic acid (the functional monomer) and ethylene glycol dimethacrylate (the crosslinker) in the presence of quercetin (the template) on the ITO. The template was extracted, and the remaining ITO electrode used for the amperometric determination of quercetin at a working potential of 0.26 V (vs. SCE). The analytical range is from 5.10^?8 to 10^?4 mol L^?1, and the detection limit is 5.10^?8 mol L^?1.

Syntheses of novel calix[4]arene hydrazone-based receptors and their cooperative complexation with soft and hard metal ions

Four novel calix[4]arene hydrazone-based receptors 3a?Cd were prepared in yields of 69?C87% by condensating formylated calix[4]arene ester (2) with salicylyl hydrazine, 2,4-dinitrophenyl hydrazine, nicotinyl hydrazine or phenyl thiosemicarhazide, respectively. New compounds were characterized through elemental analysis, IR, ESI?CMS, ¹H NMR studies. Compounds 3a?Cd containing two binding sites had the complexation abilities for hard and soft cations concurrently. The noncompetitive extracting experiments showed compounds 3a?Cd were excellent receptors for hard and soft metal cations. The competitive extracting experiments exhibited the cooperative complexation in binding hard and soft metal cations and compound 3a possessed outstanding selectivity for Na⁺ and Hg²⁺. The IR spectra of compound 3a before and after complexation revealed that the soft metal cation was binded in the cavity composed of hydrazone groups and azo groups at the upper rims of calix[4]arene units and hard metal cations was binded in cavity composed of ester groups and phenolic hydroxyl groups at the lower rims of calix[4]arene units.     

Alkylation of 1-hydroxy-1H-[1,2,3]triazolo[4,5-e][1,2,3,4]tetrazine 5,7-dioxide

Alkylation of 1-hydroxy-1H-[1,2,3]triazolo[4,5-e][1,2,3,4]tetrazine 5,7-dioxide 1 and its silver salt 10 with different alkylating agents (diazomethane, diazoacetone, bromoacetone, α-bromoacetophenone, methyl iodide, methyl vinyl ketone) was studied. Alkylation of compound 1 with diazo compounds and salt 10 with halocompounds results predominantly in O-alkylation products, 1-alkoxy-1H-[1,2,3]triazolo[4,5-e][1,2,3,4]tetrazine 5,7-dioxides. The Michael reaction of compound 1 with methyl vinyl ketone involves the triazole nitrogen atom to give 1-(3-oxobutyl)-1H-[1,2,3]triazolo[4,5-e][1,2,3,4]tetrazine 3,4,6-trioxide. The structures of the compounds synthesized were established by ¹H, ¹³C, ¹⁴N NMR spectroscopy and mass spectrometry.