Carbohydrate–Lipid Interactions: Affinities of Methylmannose Polysaccharides for Lipids in Aqueous Solution

The interactions between 3‐O‐methyl‐mannose polysaccharides (MMPs), extracted from Mycobacterium smegmatis (consisting of a mixture of MMP‐10, ‐11, ‐12 and ‐13) or obtained by chemical synthesis (MMP‐5_s, ‐8_s, ‐11_s and ‐14_s), and linear saturated and unsaturated fatty acids (FAs), and a commercial mixture of naphthenic acids (NAs) in aqueous solution at 25 °C and pH 8.5 were quantified by electrospray ionization mass spectrometry (ESI‐MS). Association constants (K_a) for MMP binding to four FAs (myristic acid, palmitic acid, stearic acid and trans‐parinaric acid) were measured by using an indirect ESI‐MS assay, the “proxy protein” method. The K_a values are in the 10⁴–10⁵ M ^?1 range and, based on results obtained for the binding of the synthetic MMPs with palmitic acid, increase with the size of the carbohydrate. Notably, the measured affinity of the extracted MMPs for trans‐parinaric acid is two orders of magnitude smaller than the reported value, which was determined by using a fluorescence assay. Using a newly developed competitive binding assay, referred to as the “proxy protein/proxy ligand” ESI‐MS method, it was shown that MMPs bind specifically to NAs in aqueous solution, with apparent affinities of approximately (5×10⁴) M ^?1 for the mixture of NAs tested. This represents the first demonstration that MMPs can bind to hydrophobic species more complex than those containing linear alkyl/alkenyl chains. Moreover, the approach developed here represents a novel method for probing carbohydrate–lipid interactions.     

Rapid and Quantitative Blood Analysis for Free Fatty Acids by Chemical Ionization Mass Spectrometry

Abstract

A quantitative analysis of fatty acids in micro-samples of dried blood spots by chemical ionization mass spectrometry has been developed. Isotope determination was used as the quantitating technique using the corresponding deuterium labeled internal standards. The procedure yields excellent precision and accuracy as demonstrated by the analysis of known fatty acid mixtures and of both C_12:0 to C_18:0 acids and phytanic acid in the blood from patients.     

Specificity of receptor-ligand interactions and their effect on dimerisation as observed by electrospray mass spectrometry: bile acids form stable adducts to the RXRalpha

Electrospray (ES) mass spectrometry data is presented showing that agonist binding to the nuclear receptor (NR), retinoid X receptor alpha (RXRalpha), is competitive. The competitive nature of agonist binding can be used to discriminate between the specific and non-specific binding of small lipophilic molecules to NRs. Further, data is presented which show that high-affinity ligand binding to the RXRalpha ligand-binding domain (LBD) stabilises the domain homodimer. The results indicate that homodimerisation, a functional property of the receptor associated with the binding of agonist ligands, could be used to discriminate between specific and non-specific binding events. Additionally, we report on the remarkable stability of the gas-phase complex between the RXRalpha LBD protein and endogenous bile acids. Protein-bile acid interactions in the gas phase were found to be surprisingly strong, withstanding 'in-source' fragmentation in the ES interface, and, in the case of taurocholic acid (TCA) and lithocholic acid-3-sulphate (LCA-3-sulphate), collision-induced dissociation within the collision cell of a tandem mass spectrometer. Bile acids were found to be inactive towards RXRalpha in transfection assays, and have not been reported to be ligands for the RXRalpha, although lithocholic acid (LCA) has been found to be a competitor in the photoaffinity labelling of RXRbeta with 9-cis-retinoic acid (9-cis-RA). The observation of strong RXRalpha-bile acid non-covalent complexes in ES mass spectrometry highlight the danger of extrapolating gas-phase binding data to the solution phase and further to a possible biological activity, particularly when surface-active compounds such as bile acids are involved. The introduction of a competitive ligand-binding experiment can alleviate this problem and allow the differentiation between specific and non-specific binding.     

Mass spectrometric study of organocyclosiloxanes

Organocyclosiloxanes of various chemical structures were studied by mass spectrometry using different ionization methods. The electron ionization mass spectra contain no peaks of molecular ions, and the main fragment ions are formed due to complicated rearrangements in a molecular ion, which provides no comprehensive view about the molecular structure. The desorption spectra exhibit peaks of quasimolecular and fragment ions, which characterize both molecular weights and chemical structures of the compounds under study. Dedicated to Academician G. A. Abakumov on the occasion of his 70th birthday. Published in Russian in Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 9, pp. 1746–1749, September, 2007.     

Enhancement of Ultraviolet Detectability of Fatty Acids for Purposes of Liquid Chromatographic-Mass Spectrometric Analyses

Abstract

A technique for benzyl derivatization of fatty acids which results in enhanced ultraviolet absorption with a concomittant increase in sensitivity in liquid chromatographic (LC) separations has been developed. Benzylation prior to liquid chromatographic separation also provides a uniform enhancement of response for fatty acids which permits direct relative quantization without acquisition of additional calibration data. After removal of excess solvent from eluted compounds mass spectra were determined using a direct probe which confirmed the benzyl ester structures. A discussion of spectral data and the advantages of using mass spectrometry as an ancillary tool to liquid chromatography is also discussed.     

Structural characterization of fatty acids cationized with copper by electrospray ionization mass spectrometry under low-energy collision-induced dissociation

Fatty acids have for many years been characterized by mass spectrometry using electron ionization after chemical derivatization. When fatty acids are ionized using desorption/ionization methods such as electrospray ionization or fast atom bombardment, structural information is usually obtained through high-energy collision-induced dissociation (CID) using sector instruments. It has been shown that copper displays very interesting properties in the gas phase during CID. In this study, the reactivity of saturated and unsaturated fatty acid-copper [M-H+Cu(II)]+ complex and the role of the copper ion in promoting fragmentations were investigated under low-energy collisional activation conditions. The decomposition of these species in an ion trap instrument led to diagnostic ion series that reflect C--C bond cleavage, which involves Cu(II) reduction followed by the release of an alkyl radical. It was demonstrated that in this way the localization of one or two homoconjugated double bonds is possible using low-energy CID. Moreover, the distinction of cis and trans isomers is possible through characteristic product ions related to a specific loss of CO2. When these experiments are repeated using a triple-quadrupole instrument with argon as collision gas, a different behavior is observed as in this case, in addition to the product ion distributions observed in the ion trap, other distributions are observed that reflect the influence of the different kinetic shifts and the occurrence of consecutive decompositions. Different examples are presented with various saturated and unsaturated fatty acid chains. Mechanisms are proposed in order to rationalize the experimental observations.     

Mass spectrometry based cellular phosphoinositides profiling and phospholipid analysis: A brief review

Phospholipids are key components of cellular membrane and signaling. Among cellular phospholipids, phosphoinositides, phosphorylated derivatives of phosphatidylinositol are important as a participant in essential metabolic processes in animals. However, due to its low abundance in cells and tissues, it is difficult to identify the composition of phosphoinositides. Recent advances in mass spectrometric techniques, combined with established separation methods, have allowed the rapid and sensitive detection and quantification of a variety of lipid species including phosphoinositides. In this mini review, we briefly introduce progress in profiling of cellular phosphoinositides using mass spectrometry. We also summarize current progress of matrices development for the analysis of cellular phospholipids using matrix-assisted laser desorption/ionization mass spectrometry. The phosphoinositides profiling and phospholipids imaging will help us to understand how they function in a biological system and will provide a powerful tool for elucidating the mechanism of diseases such as diabetes, cancer and neurodegenerative diseases. The investigation of cellular phospholipids including phosphoinositides using electrospray ionization mass spectrometry and matrix-assisted laser desorption/ionization mass spectrometry will suggest new insights on human diseases, and on clinical application through drug development of lipid related diseases.     

Mass spectrometric methods for the analysis of chlorinated and nitrated isoflavonoids: a novel class of biological metabolites

Electrospray ionization combined with tandem mass spectrometry was applied to a study of some representative chlorinated and nitrated isoflavones-potential metabolites of isoflavones in inflammatory cells. Upon collision-induced dissociation of deprotonated [M - H](-) ions of these compounds, a number of structurally characteristic product ions were produced. The product ion analysis of 3'- and 8-chlorodaidzein in the tandom mass spectra led to ready differentiation of these isomers. 3-Nitro derivatives of both genistein and daidzein have product ions due to the losses of HNO(2) and two OH groups. Chlorinated derivatives of isoflavones were detected in cell-based experiments and their structures were proposed by comparing the tandem mass spectra of their product ions with those of standards. This work provides a suitable analytical basis to aid the characterization of chlorinated and nitrated metabolites in studies in vivo and in vitro.     

A Chemical Ionization Mass Spectrometric Study of Fluorescamine and Fluorescamine - Amino Acid Derivatives

Abstract

The chemical ionization mass spectra of fluorescamine and fluorescamine - amino acid derivatives have been studied using methane and ammonia as reagent gases. Major ions in the spectra are protonated molecular ions, adduct ions and ions formed by loss of an oxygen atom.

Fluorescamine, 4-phenyl-spiro[furan-2(3H),1′-phthalan]3,3′-dione, is a powerful new fluorogenic reagent for assaying primary amines.¹ and EI² and EI³ mass spectrometric investigations of fluorescamine and its derivatives were carried out. Our present study reports the CI mass spectral analysis of fluorescamine and some of its amino acid derivatives.     

Mass spectrometric studies of 2-aryl-5-acetylthiazole derivatives

Electron ionisation mass spectrometry was usefully used to characterize structurally 2-aryl-5-acetylthiazole derivatives in the gas phase. The compounds show characteristic fragmentation pathways depending on the chemical nature of the substituent at position 2, consisting mainly in the cleavage of both the 1,2- and 3,4-bonds of the thiazole ring. Liquid secondary ion mass spectrometry was applied to study the effects of protonation on the gas-phase unimolecular reactions of this class of compound. Tandem mass spectrometric experiments, carried out on molecular and protonated molecular ions, and also on fragment ions produced in the source, allowed the elucidation of gas-phase decompositions of low-internal energy ions.     

取代螺环戊烷的电子轰击和化学电离正,负离子质谱

本文报道了苯取代螺环戊烷衍生物的电子轰击(EI)正离子和化学电离正、负离子(PNCI)质谱。通过亚稳离子测定,研究了该类化合物的裂解机理。在卤代螺环戊烷的EI质谱中,分子离子峰都很弱,甚至不出现M 离子。其特征离子为[M-X]~ 、[M-2X] 和[M-X-HX]~ 。CI正离子谱有较强的[M H]~ 、[M-2X]~ 和[M-x]~ ,CI负离子谱的特征离子为[M X]~-,它们在多数情况下为基峰离子,另外还出现HX_2~-或X~-离子。     

Modification of dipeptides by alkyl chloroformate—alkanol mixtures for analysis by gas chromatography/mass spectrometry with electron and chemical ionization and collisional activation: Differentiation of isomers

A possibility of dipeptide derivatization by alkyl chloroformate—alkanol (Alk = Me, Et, Pr, Bu) mixed reactants for their determination and differentiation of isomers in mixtures by gas chromatography/mass spectrometry with electron and chemical ionization and collisional activation was studied. Prospects for using derivatization by mixtures of methyl chloroformate with 2,2,2-trifluoroethanol, 2,2,3,3,3-pentafluoropropanol, and 2,2,3,3,4,4,4-heptafluorobutanol were shown. Diagnostic ions that make it possible to determine the amino acid sequence in dipeptides and distinguish residues of isomeric leucine and isoleucine were revealed. Published in Russian in Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 12, pp. 2200–2204, December, 2006.     

邻氨基苯酚化学修饰试剂用于气相色谱/质谱分析花粉脂肪酸

采用邻氨基苯酚作为脂肪酸的化学修饰试剂,将羧基修改为含氮杂环,使在EI源中避免链烯基中碳碳双键的移动。以气相色谱／EI质谱分析花粉脂肪酸,解析脂肪酸邻氨基苯酚化学修饰产物的EI质谱图;讨论了烯酸中碳碳双键的定位规则;鉴定出巨日花粉12种脂肪酸,由C12-C24脂肪酸组成,不饱和脂肪酸的含量占73．94％,其中多不饱和脂肪酸含量占47．61％,多不饱和脂肪酸与饱和脂肪酸之比达1.85,还检出了在其他花粉中极为少见的人体必需脂肪酸15-二十四烯酸(神经酸)。     

常压电喷雾离子化的机理及应用

作为最有前景的分析仪器之一,质谱技术已在药物、食品、环境、人类健康、国家安全及相关领域展现出广阔的应用前景。不同种类的分析物具有多种特征,这为直接离子化及质谱分析增加了难度。常压敞开式离子源是近年来新兴的一种离子源,这类离子源具有无需复杂的样品前处理、操作方便、快速、非破坏性、灵敏度及特异性好、能实现实时原位、高通量分析等特点。本文综述了基于电喷雾离子化(ESI)原理的各种离子源的电离机理、特征及应用,展望了常压敞开式离子源的发展趋势。