Number Of Exchangeable Protons Research Articles

Structure-performance relationships of phenyl cinnamic acid derivatives as MALDI-MS matrices for sulfatide detection.

A key aspect for the further development of matrix-assisted laser desorption ionization (MALDI)-mass spectrometry (MS) is a better understanding of the working principles of MALDI matrices. To address this issue, a chemical compound library of 59 structurally related cinnamic acid derivatives was synthesized. Potential MALDI matrices were evaluated with sulfatides, a class of anionic lipids which are abundant in complex brain lipid mixtures. For each matrix relative mean S/N ratios of sulfatides were determined against 9-aminoacridine as a reference matrix using negative ion mass spectrometry with 355 and 337nm laser systems. The comparison of matrix features with their corresponding relative mean S/N ratios for sulfatide detection identified correlations between matrix substitution patterns, their chemical functionality, and their MALDI-MS performance. Crystal structures of six selected matrices provided structural insight in hydrogen bond interactions in the solid state. Principal component analysis allowed the additional identification of correlation trends between structural and physical matrix properties like number of exchangeable protons at the head group, MW, logP, UV-Vis, and sulfatide detection sensitivity. Graphical abstract Design, synthesis and mass spectrometric evaluation of MALDI-MS matrix compound libraries allows the identification of matrix structure - MALDI-MS performance relationships using multivariate statistics as a tool.

Entrapment of a neutral Tm(III)‐based complex with two inner‐sphere coordinated water molecules into PEG‐stabilized vesicles: towards an alternative strategy to develop high‐performance LipoCEST contrast agents for MR imaging

Chemical exchange saturation transfer (CEST) probes issued from the encapsulation of a water proton paramagnetic shift reagent into the inner aqueous volume of lipid vesicles provide an emerging class of frequency-selective contrast agents with huge potential in the field of molecular magnetic resonance imaging (MRI). This work deals with the generation of such LipoCEST agents properly designed to optimize, under isotonic conditions, the chemical shift offset of the intra-liposomal water protons as well as the number of exchangeable protons under reasonably low radiofrequency (RF) fields of saturation. The strategy lies in the loading of poly(ethylene glycol)-stabilized nanosized liposomes with uncharged lanthanide chelates, binding more than one water molecule in the first hydration sphere, exemplified here by [Tm(III)-DO3A (H2 O)2 ] complex. The key properties of the probes are demonstrated by complementary NMR investigations. The residence lifetime of the water molecules coordinated to the lanthanide center was outstandingly short (9.5 ± 0.2 ns from (17) O NMR), and indeed relevant for effective LipoCEST responsiveness. The (1) H NMR CEST spectra (7.01 T magnetic field) prove that the theoretically expected optimal sensitivity can be approximated in the nanomolar concentration range, at reasonably low RF presaturation pulses (6.7-12 μT) and saturation frequency offsets of the intra-liposomal water protons beyond 10 ppm, making possible selective irradiation in biological environment. CEST-MRI images (7.01 T magnetic field and 10-12 μT RF pulse) explicitly confirm the interest of these newly conceived LipoCEST agents, indeed among the most efficient ones developed so far under isosmotic conditions.

Identification of Regions of Rabbit Muscle Pyruvate Kinase Important for Allosteric Regulation by Phenylalanine, Detected by H/D Exchange Mass Spectrometry

Mass spectrometry has been used to determine the number of exchangeable backbone amide protons and the associated rate constants that are altered when rabbit muscle pyruvate kinase (rM1-PYK) binds either the allosteric inhibitor (phenylalanine) or a nonallosteric analogue of the inhibitor. Alanine is used as the nonallosteric analogue because it binds competitively with phenylalanine but elicits a negligible allosteric inhibition, i.e., a negligible reduction in the affinity of rM1-PYK for the substrate, phosphoenolpyruvate. This experimental design is expected to distinguish changes in the protein caused by effector binding (i.e., those changes common upon the addition of alanine vs phenylalanine) from changes associated with allosteric regulation (i.e., those elicited by the addition of phenylalanine binding, but not alanine binding). High-quality peptic fragments covering 98% of the protein were identified. Changes in both the number of exchangeable protons per peptide and in the rate constant associated with exchange highlight regions of the protein with allosteric roles. The set of allosterically relevant peptides identified by this technique includes residues previously identified by mutagenesis to have roles in allosteric regulation by phenylalanine.

A simple method for differentiation of monoisotopic drug metabolites with hydrogen–deuterium exchange liquid chromatography/electrospray mass spectrometry

A simple but efficient method for determination of labile protons in drug metabolites using post-column infusion of deuterium oxide (D 2O) in liquid chromatography/mass spectrometry (LC/MS) experiments with electrospray ionization and time-of-flight mass spectrometry is described. The number of exchangeable protons in analytes, i.e. hydroxyl, amine, thiol and carboxylic acid protons, can easily be determined by comparing the increase in m/ z values after H/D-exchange occurring online between a HPLC column and electrospray ion source. Especially, the hydroxyl metabolites and S/N-oxides with the same accurate mass can be distinguished. A good degree of exchange was obtained in repeatable experiments. Only a low consumption of D 2O is needed in a very easy and rapidly set-up procedure. The method is applied in the study of metabolites of omeprazole and imipramine in human and mouse in vitro samples, respectively, together with exact mass data obtained from time-of-flight mass spectrometric experiments.

質量分析法を用いたタンパク質の揺らぎの研究

Protein dynamics is a basis for understanding the structure-function relationships of proteins, then the magnitude and time scale of fluctuation have been widely investigated by means of various spectroscopic and relaxation methods: B-factor of X-ray crystallography, order parameter of NMR, and compressibility, etc. Among them, hydrogen/deuterium (H/D) exchange kinetics is a novel technique, which gives important information on the proton exchange rate and the number of exchangeable protons. H/D exchange of proteins has been mainly monitored by IR and NMR, but mass spectrometry has been recently utilized in this area. Mass spectrometry has various advantages: the mass change by H/D exchange can be measured with an accuracy of 1 Da using only small amount of sample even if it is a mixture involving some conformers. In this paper, we briefly introduce our method to study the H/D exchange kinetics using a MALDI-TOF-MS coupled with pepsin digestion.

Synthesis, Characterization and lithium Intercalation behavior of electrodeposited V2O5

Vanadium pentoxide deposits have been prepared by electrochemical oxidation of vanadyl ions in aqueous solution. They were characterized with the use of a combination of techniques including: X-ray diffraction, TGA, SEM, chemical titration… They exhibit a 2D structure similar to that of V2O5 gels and contain the same number of exchangeable protons and water molecules per formula unit. However clear differences are also observed, mainly related to the V4+ content, the interlayer distance and their evolution with thermal treatments. The electrochemical behavior of as prepared and heat treated electrodeposited materials was investigated with respect to lithium intercalation. A thermal treatment at T>140°C induces an irreversible decrease of the V4+ content and leads to a higher specific capacity.

Identification of amperozide metabolites in urine from rats, rabbits, dogs and man by Frit‐FAB LC/MS using deuterated solvents to gain additional structural information

AbstractA general procedure for screening of amperozide, N‐ethyl‐4‐[4,4‐bis(p‐fluorophenylbutyl)]‐1‐piperazine carboxamide, labelled with 3H and 14C was developed. Urine extracts were first fractionated by preparative reversedphase chromatography with an acetonitrile gradient elution. The collected fractions were finally analysed using a methanol gradient on packed capillary LC columns with an internal diameter of 0.32 or 0.5 mm connected to the Frit‐FAB probe of a Jeol SX‐102 mass spectrometer for structural analysis. A micro‐gradient system dedicated for the use of deuterated solvents was constructed from two six‐port switching valves to reduce the consumption of the eluents. The number of hydrogens bound to heteroatoms (OH, NH) was determined by comparing the spectra recorded from mobile phases using water and deuterium oxide. The mass spectra recorded during elution with deuterated solvents was also useful for the interpretation of the fragmentation pattern of standard compounds and unknown metabolites. The technique proved especially useful to differentiate between hydroxylation and N‐oxidation which gave the same increase in molecular mass by 16 u but a difference in the number of exchangeable protons. Metabolites formed by oxidative N‐dealkylation of amperozide either at the basic nitrogen or at the N‐ethylcarboxamide nitrogen were identified. Additional metabolites derived from deethylated amperozide inovlving N‐oxidation of the basic nitrogen of the piperazine ring and/or hydroxylation of the piperazine ring were identified. Metabolites formed by oxidative N‐dealkylation and opening of the piperazine ring were also identified.

Biomolecular folding in vacuo!!!(?)

Biomolecular folding in vacuo!!!(?)

Applications of electrospray mass spectrometry to studies on the structural properties of ribonuclease A and ribonuclease B

AbstractDeuterium‐exchange experiments were performed on ribonuclease A (RNase A) and its glycosylated form, ribonuclease B (RNase B), using electrospray ionization mass spectrometry. The number of exchangeable protons was found to be similar for both forms of the enzyme, indicating that the oligosaccharide moiety on RNase B does not exert any influence on protein conformation. Four main glycoform populations were identified in RNase B, each of which was homogeneous and associated with one phosphate group. Spectra for RNase A were heterogeneous due to the presence of 0–3 phosphate groups bound to the enzyme.

Ammonia (NH3 and N2H3) direct chemical ionization mass spectrometry of underivatized prostaglandin-H2 and other selected stable prostaglandins.

A study of the positive and negative ion, ammonia (NH3 and N2H3) direct chemical ionization mass spectrometry of highly purified prostaglandin endoperoxide (PGH2) is presented. The positive ion spectra were characterized by an intense [M + NH4]+ adduct at m/z 370 and several fragment ions, most notably a [M + NH4-H2O]+ ion at m/z 352 and an ion at m/z 298, assigned as the [M + NH4-72]+ ion of 12-hydroxy-5,8,10-heptadecatrienoic acid formed form PGH2 in the spectrometer. The negative ion spectra of PGH2 were characterized by a base peak at m/z 352 [M]- and by an ion at m/z 334 corresponding to the loss of water from the parent ion. A combination of negative ion and deuterated ammonia reagent gas was used in making assignments and in demonstrating that the spectra observed were due to intact PGH2 and its stable PGE2 and PGD2 isomers formed in the spectrometer. In addition, use of the latter reagent gas was shown to clearly distinguish between several arachidonic acid metabolites, differing in their number of exchangeable protons. Furthermore, preliminary results with several stable prostaglandins indicate that the spectra are sensitive to different functional groups that are present. Consequently, it would appear that negative and positive ion, NH3 (N2H3) direct chemical ionization mass spectrometry would be useful in the analysis of labile arachidonic acid metabolites, without the need for prior derivatization.