Françoise Fournier

Biotin synthase mechanism: on the origin of sulphur

Biotin synthase catalyses the last step of the biosynthesis of biotin in microorganisms and plants. The active protein isolated from Bacillus sphaericus and Escherichia coli contains an iron‐sulphur (FeS) cluster. The native enzymes were depleted of their iron and inorganic sulphide and the resulting apoenzymes were chemically reconstituted with FeCl3 and Na2[34S] to give labelled (Fe34S) enzymes. These enzymes were functional and when assayed in vitro produced labelled biotin containing about 65% of 34S. These data strongly support the hypothesis that the sulphur of biotin is derived from the (FeS) centre of the enzyme.

Characterization of the ceramide moieties of sphingoglycolipids from mouse brain by ESI-MS/MS identification of ceramides containing sphingadienine

Sphingoglycolipids (SGLs) are cell membrane constituents. As the ceramide structure influences the biological properties of the SGL, we characterized by electrospray ionization tandem mass spectrometry the molecular species of ceramides present in SGL of mouse brain. We report here for the first time the presence in mammalian brain of sphingadienine (d18:2). Sphingenine (d18:1) is present in all SGL species, in contrast to eicosasphingenine (d20:1), which is a constituent of only gangliosides. Sphingadienine is present in galactosylceramide and sulfatides. Free ceramides contain the three types of bases. Thus, there could be two separate pools of free ceramides (d18:1, d18:2 and d20:1, d18:1) as precursors of complex SGL.

Proton affinities of the commonly occuring L-amino acids by using electrospray ionization-ion trap mass spectrometry

In this study, the use of an electrospray ionization (ESI)-ion trap mass spectrometry to perform thermochemical determinations using the kinetic method is shown. In this method, competitive dissociations of selected proton-bound heterodimeric [BiHB]+ ions are investigated and particularly, the product ion abundance [BiH+] / [BH+] ratio is accurately measured and compared to the proton affinity of each neutral partner (i.e. the Bi and B). The proton affinities of the amino acids are well known and these compounds were chosen to investigate the ion trap potentiality for such purpose, particularly, when externally prepared ions were injected. It appears that, in spite of the low abundance of homo and heterodimer ions, constant and reproducible [BiH+] / [BH+] ratios are obtained when sequential tandem mass spectrometry (MS/MS) experiments are performed within similar ion trapping conditions. Such conditions are reached for a constant ion excitation qz value. The resulting ln([BiH+] / [BH+]) dependence upon the PABi and PAB values (from the literature) is linear and yields a reproducible slope after several measurements are made to determine the accuracy of this approach. Under various excitation conditions, in spite of the change in effective temperature, neither the scale order nor the PA values are changed. Furthermore, entropy variation (δΔS0) between leucine and other amino acids was evaluated by several methods.

Zinc binding properties of the amyloid fragment Aβ(1–16) studied by electrospray-ionization mass spectrometry

A major hallmark of Alzheimer’s disease (AD) is the strong accumulation in brain of senile plaques, mainly composed of the amyloid-β peptide (Aβ). Recent studies have suggested that the zinc cation would be a possible key mediating factor for the formation of amyloid extracellular deposits, by binding to Aβ and triggering the involved aggregation process. From a previous circular dichroism (CD) study, we have proposed the N-terminal 1–16 region of Aβ(1–16), as the minimal fragment able to specifically bind zinc. Here we investigate the Zn2+ binding properties of Aβ(1–16) by electrospray-ionization mass spectrometry (ESI-MS). The stoichiometry of Aβ(1–16)/Zn2+ association and the relative affinity of different cations towards Aβ(1–16) are investigated by analyzing the mass spectra of Aβ(1–16) in the presence of different cations, introduced alone or in competition. Zn2+ binding sites are determined from collision-induced dissociation (CID) experiments conducted on the Aβ(1–16) cationized species. From these data, Aβ(1–16) is shown to form a 1:1 complex with Zn2+ and to bind up to three cations upon increasing the Zn2+ concentration. Under CID, zinc binding induces specific cleavages after the three histidines of the Aβ(1–16) sequence (H6, H13 and H14), showing their simultaneous implication in the Zn2+ coordination sphere. The binding of Aβ(1–16) to several Zn2+ cations appears less specific, but still implicates the three histidines, each of them behaving thus as an autonomous binding site. A model is proposed to explain both the specific and the aspecific interactions of Zn2+ with Aβ(1–16) that is confirmed here to behave as the minimal zinc-binding region of Aβ.

Endogenous Xenopus-oocyte Ca-channels are regulated by protein kinases A and C

Calcium entry into Xenopus oocyte occurs mainly through voltage‐dependent calcium channels. These channels were characterized as belonging to a particular type of calcium channel insensitive to dihydropyridines, ω‐conotoxin, and Agelenopsis aperta venom, but blocked by divalent cations (Co, Cd, Ni). Intracellular injection of cAMP, or bath application of phorbol ester, induced a marked increase in calcium current amplitude and a slowing of the inactivation time‐course. Despite their different pharmacology, endogenous calcium channels, like cardiac or neuronal calcium channels, could be thus regulated by protein kinases A and C.

Stereochemical differentiation of four mono-saccharides using transition metal complexes by electrospray ionization/ion-trap mass spectrometry

Several approaches may contribute to the structural elucidation of carbohydrates. The distinction of diastereoisomers by mass spectrometry is often a tedious task and, ultimately, the stereochemistry of monosaccharides must be determined. α/β anomers of glucose, mannose, galactose and talose stereochemically differ at the C2 and C4 positions and this work describes an efficient system used to differentiate four isomeric monosaccharides easily. The approach is based on mass spectrometry and it uses the gas-phase interaction between a sugar molecule and the iron(II) transition metal ion for differentiation of the species. Electrospray ionization allows the generation of cationized [M + FeCl]+ monosaccharides. Such ions have been prepared in high abundance in the external source of an electrospray ionization/ion-trap mass spectrometer instrument. Resonant excitation of these selected ions gives rise to dissociative collisions. Very likely, the strong interaction of ironII with nucleophilic sites (i.e. hydroxyl groups) seems to favor regioselective and stereoselective ironII ion attachment to each epimer. Consequently, when submitted to resonant excitation conditions, the cluster [M + FeCl]+ ions undergo fragmentations that can either be stereospecific in certain cases or, more usually, kinetically orientated (i.e. stereochemical effects on the rate constants of competitive unimolecular processes). Thus, by scrutinizing the variation in the relative abundances of diagnostic fragment ions, it is possible to distinguish stereoisomeric sugars. The origin of some product ions was proved by MS2 and MS n experiments through 13C and 2H-labeling that may enlighten the mechanisms.

Gas-phase ionization/desolvation processes and their effect on protein charge state distribution under matrix-assisted laser desorption/ionization conditions.

The charge state distribution of proteins was studied as a function of experimental conditions, to improve the understanding of the matrix-assisted laser desorption/ionization (MALDI) mechanisms. The relative abundances of the multiply-charged ions appear to be a function of the matrix chosen, the laser fluence and the matrix-to-analyte molar ratio. A correlation is found between the matrix proton affinity and the yield of singly- versus multiply-charged ions. These results are in good agreement with a model in which gas-phase intracluster reactions play a significant role in analyte ion formation. A new model for endothermic desolvation processes in ultraviolet/MALDI is presented and discussed. It is based upon the existence of highly-charged precursor clusters and, complementary to the ion survivor model of Karas et al., assumes that two energy-dependent processes exist: (i) a soft desolvation involving consecutive losses of neutral matrix molecules, leading to a multiply-charged analyte and (ii) hard desolvation leading to a low charge state analyte, by consecutive losses of charged matrix molecules. These desolvations pathways are discussed in terms of kinetically limited processes. The efficiency of the two competitive desolvation processes seems related to the internal energy carried away by clusters during ablation.

Can Cluster Structure Affect Kinetic Method Measurements? The Curious Case of Glutamic Acid's Gas-Phase Acidity

The gas-phase acidities of aspartic, glutamic, and 2-aminoadipic acid have been determined by the kinetic method in a triple-quadrupole instrument. Although aspartic acid behaves in the conventional way and gives a ΔHacid value of 1340 kJ mol−1, glutamic and 2-aminoadipic acids give kinetic method plots with two distinct slopes. This leads to ΔHacid values of 1350 and 1366 kJ mol−1 for glutamic acid, and 1355 and 1369 kJ mol−1 for 2-aminoadipic acid. The value for aspartic acid and the low collision energy value for glutamic acid are consistent with recent measurements by Poutsma and co-workers in a quadrupole ion trap. The experiments are supported by calculations at the G3(MP2) and OLYP/aug-cc-pVTZ levels. Computational studies of model clusters of the amino acids with trifluoroacetate suggest there are distinct preferences. Glutamic and 2-aminoadipic acid prefer clusters where the amino acid adopts a zwitterion-like structure whereas aspartic acid prefers to adopt a conventional (canonic) structure in its clusters. This result along with the computed stabilities of zwitterion-like conformations of the deprotonated amino acids leads to the following explanation for the presence of two slopes in the kinetic method plots. At low collision energies, the deprotonated amino acid dissociates from the cluster, with rearrangement if necessary, to give its preferred conformation, but at high collision energies, the deprotonated amino acid directly dissociates in the conformation preferred in the cluster. For glutamic and 2-aminoadipic acids, this is a zwitterion-like structure that is about 20 kJ mol−1 less stable than the global minimum.

Stereochemical effects from doubly-charged iron clusters for the structural elucidation of diastereomeric monosaccharides using ESI/IT-MS

Abstract Several approaches may be used to contribute to the structural elucidation of carbohydrates. Distinction of diastereomeric monosaccharides is often a tedious task by mass spectrometry, and ultimately the monosaccharide stereochemistry must be determined. Diastereomers of glucose, mannose, galactose and talose stereochemically differ at the C (2) and C (4) positions and previous work described an efficient system used to differentiate these four monosaccharides easily. The approach was based on ESI mass spectrometry and scrutinized the behavior of a sugar molecule towards the iron(II) chloride and the resulting effects in the gas phase. The produced cationized monomeric [MFe II Cl] + and dimeric [M 2 Fe II Cl] + ions as singly-charged species were studied by sequential MS/MS experiments. When submitted to low resonant excitation conditions these selected ions undergo fragmentation within a kinetic control (i.e., stereochemical effects on the rate constants of competitive unimolecular processes). Enhancement of stereochemical effects was displayed in the CID spectra of the cationized [M 2 Fe II Cl] + dimeric ions. The present work first reports the influence of the sample preparation on the produced cationized species using the electrospray process. Formation of doubly-charged cluster M n Fe 2+ ions when the sugar molecule (M) is introduced in excess with the iron(II) chloride was observed. Secondly, behavior of these cluster ions under resonant excitation conditions reveals an increase of stereochemical effects compared with that observed in the case of singly-charged monomeric [MFe II Cl] + and dimeric [M 2 Fe II Cl] + ions previously studied [Eur J Mass Spectrom 6 (2000) 421; Eur J Mass Spectrom 7 (2001) 331]. The dissociation pathways of doubly-charged complexes appeared to depend upon the n number of monosaccharides which constitute the formal ligand of the iron(II). M n Fe 2+ cluster ions are useful to efficiently determine the stereochemistry of the investigated monosaccharides and represent an advantageous alternative for the structural elucidation of their respective stereochemistry.

Stereochemical Effects During [M − H]− Dissociations of Epimeric 11-OH-17β-Estradiols and Distant Electronic Effects of Substituents at C(11) Position on Gas Phase Acidity

The affinity of estradiol derivatives for the estrogen receptor (ER) depends strongly on nature and stereochemistry of substituents in C(11) position of the 17β-estradiol (I). In this work, the stereochemistry effects of the 11α-OH-17β-estradiol (IIIα) and 11β-OH-17β-estradiol (IIIβ) were investigated using CID experiments and gas-phase acidity (ΔHacid∘) determination. The CID experiments showed that the steroids decompose via different pathways involving competitive dissociations with rate constants depending upon the α/β C(11) stereochemistry. It was shown that the fragmentations of both deprotonated [IIIα-H]− and [IIIβ-H]− epimers were initiated by the deprotonation of the most acidic site, i.e. the phenolic hydroxyl at C(3). This view was confirmed by H/D exchange and double resonance experiments. Furthermore, the ΔHacid∘ of both epimers (IIIα and IIIβ), 17β-estradiol (I), and 17-desoxyestradiol (II) was determined using the extended Cooks’ kinetic method. The resulting values allowed us to classify steroids as a function of their gas-phase acidity as follows: (IIIβ)≫(II)>(I)>(IIIα). Interestingly, the α/β C(11) stereochemistry appeared to influence strongly the gas-phase acidity. This phenomenon could be explained through stereospecific proton interaction with π-orbital cloud of A ring, which was confirmed by theoretical calculation.