Pirjo Vainiotalo

Electrospray mass and tandem mass spectrometry identification of ozone oxidation products of amino acids and small peptides

Aqueous ozonation of the 22 most common amino acids and some small peptides were studied by electrospray mass (ESI-MS) and tandem mass spectrometry. After 5 min of ozonation only His, Met, Trp, and Tyr form oxidation products clearly detectable by ESI-MS. For His, the main oxidation product is formed by the addition of three oxygen atoms, His + 3O; for Met and Tyr by the addition of one oxygen atom, Met + O and Tyr + O, and for Trp by the addition of two oxygen atoms, Trp + 2O. Ozone oxidation occurs rapidly, products are already detected after 30 s of ozonation, and the reactivity order is Met > Trp > Tyr > His. The structures of the oxygen addition products were investigated by electrospray product ion mass spectra, and by comparing these spectra to those of protonated intact amino acids, and when available, to those of model compounds. His + 3O was assigned as 2-amino-4-oxo-4-(3-formylureido)butanoic acid (1) formed by oxidation of the His imidazole ring, Met + O as methionine sulfoxide (2), Trp + 2O as N-formylkynurenine (4), and Tyr + O as a mixture of dihydroxyphenylalanines (7 and 8). Ozonation of peptides show that the same number of oxygen atoms are added as expected from the ozonation of the free amino acids. The product ion mass spectra of both the protonated intact peptides, MH+, and the main ozonation products (M + nO)H+ (n = 1–3) revealed b and y type ions as the main fragments, which allow one to assign the type and location of modified amino acid in the model peptides.

Supramolecular Sensing with Phosphonate Cavitands

Phosphonate cavitands are an emerging class of synthetic receptors for supramolecular sensing. The molecular recognition properties of the third-generation tetraphosphonate cavitands toward alcohols and water at the gas-solid interface have been evaluated by means of three complementary techniques and compared to those of the parent mono- and diphosphonate cavitands. The combined use of ESI-MS and X-ray crystallography defined precisely the host-guest association at the interface in terms of type, number, strength, and geometry of interactions. Quartz crystal microbalance (QCM) measurements then validated the predictive value of such information for sensing applications. The importance of energetically equivalent multiple interactions on sensor selectivity and sensitivity has been demonstrated by comparing the molecular recognition properties of tetraphosphonate cavitands with those of their mono- and diphosphonate counterparts.

Synthesis and anti-inflammatory effects of a series of novel 7-hydroxycoumarin derivatives.

A number of 7-hydroxycoumarins have been synthesised by Pechmann cyclisation using differently substituted resorcinols employing perchloric acid as the condensing agent. All the compounds have been characterised by analytical and spectroscopic methods. The anti-inflammatory properties were tested with LPS-induced inflammation in J774 macrophages. Expression of iNOS and COX-2 was determined by Western blot, NO by nitrite assay and IL-6 by ELISA analyses. Fifteen of the tested 7-hydroxycoumarins also inhibited IL-6 production but none of them had any major inhibitory effect on COX-2 expression.

Effect of Glycosylation and Additional Domains on the Thermostability of a Family 10 Xylanase Produced by Thermopolyspora flexuosa

ABSTRACT The effects of different structural features on the thermostability of Thermopolyspora flexuosa xylanase XYN10A were investigated. A C-terminal carbohydrate binding module had only a slight effect, whereas a polyhistidine tag increased the thermostability of XYN10A xylanase. In contrast, glycosylation at Asn26, located in an exposed loop, decreased the thermostability of the xylanase. The presence of a substrate increased stability mainly at low pH.

Ammonium ion mediated resorcarene capsules: ESI-FTICRMS study on gas-phase structure and ammonium ion affinity of tetraethyl resorcarene and its per-methylated derivative.

The ammonium ion binding affinities of tetraethyl resorcarene (1) and its per-methylated derivative (2) were studied by electrospray ionization Fourier transform ion cyclotron resonance (ESI-FTICR) mass spectrometry. Ten different ammonium ions were tested as guests for the resorcarenes. A strong tendency for complex formation was observed with all ammonium ions of size and charge distribution suitable for noncovalent interactions with the cavities of the resorcarene hosts 1 and 2. Although differences in ammonium ion affinities were observed between 1 and 2 due to the dissimilar conformations, the overall tendency was that increase in the degree of substitution and the length of carbon chain of the ammonium cation facilitated the complex formation until the sterical hindrance impeded the complexation. Dimeric as well as monomeric ammonium ion complexes were formed with resorcarene 1, but resorcarene 2 was unable to form the dimeric capsules because of the lack of H-bond donor possibilities. The nature of binding of the guest was further investigated with ion-molecule reactions and by determination of the single crystal X-ray structure of host 1 complexed with tetramethyl ammonium bromide.

Gas-Phase Ring-Chain Tautomerism in 1,3-oxazolidines

Abstract Gas-phase ring-chain tautomeric equilibria with fourteen 1,3-oxazolidines derived from norephedrine and norpseudoephedrine were studied by means of mass spectrometry. Using 14 eV ionizing electrons these equilibria were comparable to those in non-polar solvent and obeyed the simple equation: log Kx = ρ σ+ + c, where ρ = 0.58±0.06 and 0.55+0.03 and c = 0.14+0.05 and 0.30±0.03 for norephedrine and norpseudoephedrine derivatives, respectively. Approximate values of enthalpy differences were also observed.

Alkali metal mediated resorcarene capsules: An ESI-FTICRMS study on gas-phase structure and cation binding of tetraethyl resorcarene and its per-methylated derivative

Electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICRMS) with additional ab initio calculations were used to examine the alkali metal cation binding selectivity (i.e., molecular recognition) and host properties of tetraethyl resorcarene (1) and its per-methylated derivative (2). The significance of intramolecular hydrogen bonding for the crown conformation was demonstrated. The presence of intramolecular flip-flop hydrogen bonding in 1 was confirmed both with calculations and in ND3-exchange experiments. All the alkali metal cations formed host—guest complexes by docking inside the cavity of the host. Complexation with the larger cations, especially Cs+, was favored. All the alkali metal cations also formed dimeric resorcarene capsules with 1. The capsules were directly H-bonded species, with no linking solvent molecules. ND3-exchange experiments and molecular modeling revealed the significance of direct intermolecular H-bonding for the crown conformation of 1 and stability of the capsule structure.

Bradavidin II from Bradyrhizobium japonicum: a new avidin-like biotin-binding protein.

A gene encoding an avidin-like protein was discovered in the genome of B. japonicum. The gene was cloned to an expression vector and a protein, named bradavidin II, was produced in E. coli. Bradavidin II has an identity of 20-30% and a similarity of 30-40% with previously discovered bradavidin and other avidin-like proteins. It has biochemical characteristics close to those of avidin and streptavidin and binds biotin tightly. In contrast to other tetrameric avidin-like proteins studied to date, bradavidin II has no tryptophan analogous to the W110 in avidin (W120 in streptavidin), thought to be one of the most essential residues for tight biotin-binding. Homology modeling suggests that a proline residue may function analogously to tryptophan in this particular position. Structural elements of bradavidin II such as an interface residue pattern or biotin contact residues could be used as such or transferred to engineered avidin forms to improve or create new tools for biotechnological applications.

Differentiation of stereoisomeric diols by using CH3OB+OCH3 in a small Fourier transform ion cyclotron resonance mass spectrometer

Abstract Gas-phase reactions of stereoisomeric cyclic diols with CH3OB+OCH3 were examined in a small Fourier transform ion cyclotron resonance mass spectrometer. CH3OB+OCH3 is a strong electrophile and rapidly abstracts an OH group from the diols studied. This very exothermic reaction is followed by spontaneous fragmentation of the resulting ion. In addition to this reaction, cis-diols also react with CH3OB+OCH3 by an intramolecular displacement of CH3OH in the initially formed, short-lived adduct ion. The product distributions allow distinction between the cis- and trans-isomers of 1,2-cyclopentanediol, and between the cis-(diendo- and diexo-) and trans-isomers of 2,3-trinorbornanediol.

Determination of thioxylo‐oligosaccharide binding to family 11 xylanases using electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry and X‐ray crystallography

Noncovalent binding of thioxylo‐oligosaccharide inhibitors, methyl 4‐thio‐α‐xylobioside (S‐Xyl2‐Me), methyl 4,4II‐dithio‐α‐xylotrioside (S‐Xyl3‐Me), methyl 4,4II,4III‐trithio‐α‐xylotetroside (S‐Xyl4‐Me), and methyl 4,4II,4III,4IV‐tetrathio‐α‐xylopentoside (S‐Xyl5‐Me), to three family 11 endo‐1,4‐β‐xylanases from Trichoderma reesei (TRX I and TRX II) and Chaetomium thermophilum (CTX) was characterized using electrospray ionization Fourier transform ion cyclotron resonance (FT‐ICR) MS and X‐ray crystallography. Ultra‐high mass‐resolving power and mass accuracy inherent to FT‐ICR allowed mass measurements for noncovalent complexes to within |ΔM|average of 2 p.p.m. The binding constants determined by MS titration experiments were in the range 104−103 M−1, decreasing in the series of S‐Xyl5‐Me ≥ S‐Xyl4‐Me > S‐Xyl3‐Me. In contrast, S‐Xyl2‐Me did not bind to any xylanase at the initial concentration of 5–200 µm, indicating increasing affinity with increasing number of xylopyranosyl units, with a minimum requirement of three. The crystal structures of CTX–inhibitor complexes gave interesting insights into the binding. Surprisingly, none of the inhibitors occupied any of the aglycone subsites of the active site. The binding to only the glycone subsites is nonproductive for catalysis, and yet this has also been observed for other family 11 xylanases in complex with β‐d‐xylotetraose [Wakarchuk WW, Campbell RL, Sung WL, Davoodi J & Makoto Y (1994) Protein Sci3, 465–475, and Sabini E, Wilson KS, Danielsen S, Schülein M & Davies GJ (2001) Acta CrystallogrD57, 1344–1347]. Therefore, the role of the aglycone subsites remains controversial despite their obvious contribution to catalysis.