Positive-negative, chemical-ionization, direct exposure mass spectrometry of underivatized prostaglandins

Nine underivatized prostaglandins were examined using direct exposure, ammonia, chemical-ionization, pulsed positive-negative ion mass spectrometry. The positive ion spectra were characterized by (M+18)⁺ ion adducts. The negative ion spectra were characterized by ions which dependence upon the functionality present in the cyclopentane ring system (acetal for TXB₂). The E and D series prostaglandins gave (M-18)⁻ as the major negative ion, while the F series and TXB₂ were characterized by negative ions corresponding to (M-1)⁻, and PGA₂ by the parent (M)⁻ ion. Prostaglandin 6-keto-PGF_1α was anomalous in this respect showing apparent dehydration, interpreted as an overall (M-18+1)⁺ and (M-18-1)⁻ in the positive and negative ion spectra, respectively. All major ion types were shown to give essentially a linear response with respect to concentration in the 10–1000 ng range. Although these initial studies were conducted under ideal conditions, it would appear that direct chemical ionization techniques show promise for providing direct structural information on prostaglandins without the need for prior chemical derivation.     

Thermospray HPLC/MS: a new mass spectrometric technique for the profiling of steroids

The analysis of various steroid classes by thermospray HPLC-MS using solvent systems containing 0.1 M ammonium acetate has been described. For simple unconjugated 3-oxo-4-ene steroids the positive ion spectra are dominated by a parent ion M + H+ and with increasing numbers of hydroxyl group intense ions formed by sequential losses of water (M + H- n18)+ become important. Steroids with dihydroxyacetone side-chains readily lose these side-chains and the resulting (M + H-60)+ fragment is the base peak in their spectra. The (M + H-60)+ ion is not important for most steroids with glycerol-type side-chains. Although competition between thermal degradation and vaporization was observed at lower concentrations, the effect was minimized after optimizing conditions and the protonated molecular ion was easily detected when as little as 1-10 pmol of material were injected on-column. Steroid glucuronides when analyzed in the negative ion mode give simple spectra with base peak and parent ion (M-H)-. Lack of fragmentation permits facile and sensitive measurement of individual glucoronides by selected-ion-monitoring. Extensive fragmentation is seen in the positive ion mode with sequential losses of H2O from the molecular ions (M + NH4)+ and from the aglycone fragment ion. For simple unconjugated steroids the sensitivity of HPLC-MS in selected-ion-monitoring mode can be excellent. When the protonated molecular ion of testosterone was monitored the signal/noise ratio for 30 pg testosterone was about 10.     

Analysis of platelet-activating factor by GC-MS after direct derivatization with pentafluorobenzoyl chloride and heptafluorobutyric anhydride

Parallel analysis of platelet-activating factor (PAF) using chemical ionization gas chromatography-mass spectrometry after direct derivatization with pentafluorobenzoyl chloride (PFB) and heptafluorobutyric anhydride (HFB) provides a facile and highly sensitive means for detecting and elucidating the structure of the numerous alkyl-chain homologs of this acetylated phospholipid autacoid. In the present study, the PFB derivative was used for initial electron capture negative ion chemical ionization analysis of PAF candidate molecules in human PMN extracts of unknown composition. Subsequent pulsed positive ion/electron capture negative ion chemical ionization evaluation of the HFB derivative furnished a measure of the molecular weight from [MH]+ and yielded the required structural information from characteristic negative ions, in particular [M-(2HF + ketene)]- and [M-(HF + acetic acid)]-. These procedures easily permitted confirmation of the presence of C16:0-, C17:0-, C18:0-, and C18:1-AGEPC (acetyl glyceryl ether phosphocholine) in extracts of stimulated human PMN and also demonstrated that the C17:0- homolog was comprised of both straight-chain and branch-chain varieties.     

Comparative study of acidic glycosphingolipids by field desorption and secondary ion mass spectrometry

Acidic glycosphingolipids were analyzed by field desorption (FD-MS) and secondary ion mass spectrometry (SI-MS) using the primary ion Xe+ with a glycerol matrix. In the analysis of underivatized gangliosides by FD-MS, the fragment corresponding to the asialo residue resulting from the cationized cluster ion (M + Na)+ was the base peak, and ions due to cleavage at the glycosidic linkages were detected, as in the neutral glycosphingolipids. In the case of sulfatide, the ceramide fragment showed the highest intensity in the spectrum. In SI-MS spectra of acidic glycosphingolipids, (M + Na)+, (M + 2Na-H)+, and (M + K)+ were continuously detected as relatively high intensity ions during analysis of gangliosides and sulfatide. Other ions were mostly similar to those obtained by FD-MS. In FD-MS spectra of permethylated gangliosides, the cationized molecular ion (M + Na)+ was the base peak, and fragment ions due to asialo gangliosides were prominent. Other peaks were hard to detect. In SI-MS, molecular ions (M + H)+ and (M + H-32)+ and other ions due to cleavage of the glycosidic linkages were clearly detected. In this case, the sensitivity was greatly improved. Ions due to the non reducing end sugars were clearly detected, because of the relatively low intensity of ion peaks due to the glycerol matrix. It is concluded that the combination with FD-MS and SI-MS is particularly useful for the determination of molecular weight, sugar sequence and ceramide structure with sample amounting to only a few micrograms order.     

Systematic analysis of desorption chemical ionization (DCI) mass spectra of nucleic acids--7

The positive ion and negative ion pyrolysis mass spectra of the herring sperm DNA have been studied using desorption chemical ionization. The positive ion desorption chemical ionization spectra have been produced with CH4, i-C4H10, NH3, HCl and Cl2; the negative ones with N2O/CH4, N2O/i-C4H10, Cl2, CCl4, HCl and via electron capture. These spectra have been compared with the electron impact ionization spectra. We have observed an important increase of sensitivity when negative ionization has replaced the positive ionization mode. The series of diagnostic ions resulting from direct chemical ionization belong to the family of base + reagent ion X [BH + X] and base + X - HX ion [B]. Their abundance has increased considerably compared to the electron impact spectra. The application of these new diagnostic ions in nucleic acid studies is interesting especially for the much higher abundance of the usually weak dG fragment ion obtained in the negative ionization mode. The dG-base segment of the DNA is the most nucleophilic centre of the whole nucleic acid and is implicated in numerous important biochemical reactions involving, for example, proteins.     

New approach for characterization of lysosulfatide by TLC, fast atom bombardment mass spectrometry and NMR spectroscopy

Thin layer chromatography of lysosulfatide showed anomalous Rf-values in contrast with such lysosphingolipids as glucopsychosine and galactopsychosine with neutral, acidic, and alkaline developing solvents. This was thought to be due to the presence of oppositely charged sulfate and amino groups in the lysosulfatide. In the negative mode of fast atom bombardment mass spectrometry, the lysosulfatide showed the pseudo molecular ion (M-H)- peak at m/z 540 and sulfate ion peak at m/z 97, whereas in the positive mode, it showed not only the pseudo molecular ion (M+H)+ peak at m/z 542, but also the major peaks of protonated psychosine at m/z 462 and fragment ions of dehydrated sphingosine at m/z 282 and 264, 13C-NMR signals of all carbons of lysosulfatide were determined by using distortionless enhancement by polarization transfer. The difference in chemical shifts of ring carbons of galactose residue between lysosulfatide and galactopsychosine was largest at C-3 (downfield shift), thereby indicating the location of the sulfate group to be at C-3 of galactose. This conclusion is supported by the 1H-NMR spectra of the lysosulfatide and galactopsychosine. Thus, the chemical structure of lysosulfatide was confirmed by fast atom bombardment mass spectrometry and 13C- and 1H-NMR spectroscopy. Furthermore, 13C-NMR signals of C-1 to C-5 of the sphingosine moiety showed significantly different chemical shifts between the lysosulfatide and galactopsychosine. These differences suggested that C-1 to C-5 of sphingosine might be influenced by intramolecular or intermolecular interaction between the sulfate group of the galactose residue and the amino group of sphingosine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Analysis of oxidative warfarin metabolites by thermospray high-performance liquid chromatography/mass spectrometry

Oxidative metabolites of the anticoagulant, warfarin [4-hydroxy-3-(3-oxo-1-phenylbutyl)-2H-1-benzopyran-2-one], produced by the actions of cytochromes P450 were analyzed by thermospray high-performance liquid chromatography/mass spectrometry. Warfarin, dehydrowarfarin, and the 6-, 7-, 8-, and 4'-hydroxy derivatives of warfarin were found to ionize well by the thermospray process in the presence of ammonium acetate. Thermospray mass spectra of these compounds were generally dominated by the protonated molecule, (M + H)+, and ions formed by the loss of water from the protonated molecule, (M + H - H2O)+. Fragment ions arising from the hydroxycoumarin, benzylhydroxycoumarin, and phenylbutanone portions of the molecules were observed, and the relative intensity of these fragment ions was greatly increased with filament ionization and application of a high repeller potential (100-130 V). Selected-ion monitoring of the (M + H)+ and (M + H - H2O)+ ions provided sensitivities for these compounds in the 2 to 10 ng range. A method employing thermospray HPLC/MS with selected-ion monitoring and internal standard quantitation for the analysis of the oxidative metabolites of warfarin is described.     

Simultaneous determination of fatty, dicarboxylic and amino acids based on derivatization with isobutyl chloroformate followed by gas chromatography-positive ion chemical ionization mass spectrometry

Gas chromatography-mass spectrometry (GC-MS) with positive ion chemical ionization (PICI) using isobutane as reagent gas was applied for analysis of isobutoxycarbonyl/isobutyl derivatives of 13 fatty, 6 dicarboxylic and 13 amino acids in a single run. For all investigated compounds (except several amino acids) the quasimolecular ions [MH](+) were registered. Asparagine underwent fragmentation via decarboxylation followed by elimination of OC(4)H(9) ([M-117](+)), whereas serine and tyrosine produced the cluster ions [M+C(4)H(9)OCO](+). Estimated detection limits were 6-250 pg in the total ion current (TIC) mode and 3-10 times lower using the selected-ion monitoring (SIM) mode.     

Metal complexes of the mycotoxins sporidesmin A and gliotoxin, investigated by electrospray ionisation mass spectrometry.

The mycotoxin sporidesmin A (spdA), responsible for the intoxication of animals, causing facial eczema, has been investigated by electrospray ionisation mass spectrometry. Protonated [spdA+H](+) and deprotonated [spdA-H](-) ions are observed in positive and negative ion modes respectively. Reduced spdA, formed by cleavage of the disulfide bond by Na[BH(4)] gives an ion [spdA+H](-), and forms ions of the type [2spdA+M](2-) with a range of divalent metal ions M(2+)=Zn(2+), Cd(2+), Hg(2+), Sn(2+) and Fe(2+). Sodium-containing analogues [2spdA+M+Na](-) are observed, particularly at high cone voltages, where they are stable towards cone voltage-induced fragmentation, indicating appreciable stability of the (spdA)(2)M system. A competition experiment between Cd(2+) and Zn(2+) demonstrates that reduced spdA has a higher affinity for Cd(2+) ions. The related gliotoxin (gtx) forms analogous [2gtx+M](2-) and [2gtx+M+Na](-) ions. The reduction and metal complexation of spdA can be monitored by (1)H NMR spectroscopy, and results in chemical shift changes for those protons adjacent to the sulfur atoms. The isolation of a polymeric cadmium-spdA complex is also reported.     

Calcium-proton and calcium-magnesium antagonisms in calmodulin: microcalorimetric and potentiometric analyses

Microcalorimetry, pH potentiometry, and direct binding studies by equilibrium dialysis or gel filtration were performed to determine the thermodynamic functions delta Ho, delta Go, and delta So guiding the interactions of Ca2+, Mg2+, and H+ with bovine brain calmodulin. At pH 7.5, Ca2+ and Mg2+ binding are both endothermic with enthalpy changes of 19.5 and 72.8 kJ X (mol of calmodulin)-1, respectively. These enthalpy changes are identical for each of the four ion-binding domains. The affinity constants also are identical with intrinsic values of 10(5) M-1 for Ca2+ and 140 M-1 for Mg2+. Ca2+ and Mg2+ do not compete for the same binding sites: at high concentrations of both ions, a calmodulin-Ca4-Mg4 species is formed with an enthalpy value of 24.4 kJ X mol-1 with respect to calmodulin-Ca4 and -28.8 kJ X mol-1 with respect to calmodulin-Mg4. Moreover, in the presence of high concentrations of Ca2+, the affinity of each of the four ion-binding domains in calmodulin for Mg2+ is decreased by a factor of 4 and vice versa, indicative of negative free-energy coupling between Ca2+ and Mg2+ binding. Protons antagonize Ca2+ and Mg2+ binding in a different manner. Ca2+-H+ antagonism is identical in each of the four Ca2+-binding domains in the pH range 7.5-5.2. Our analyses suggest that three chemical geometries, probably carboxyl-carboxylate interactions, are responsible for this antagonism with ionization constants of 10(6.2) M-1 in the metal-free protein. Mg2+-H+ antagonism also is identical for each of the Mg2+-binding sites but is qualitatively different from Ca2+-H+ antagonism.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mass spectrometry of oligosaccharides by chloride-attachment reactions: the origin of fragment loss

The direct exposure, negative chemical ionisation, chloride-attachment mass spectrometry of trehalose and sucrose gave abundant chloride-attached molecular ions. The same feature was observed when these sugars were subjected to fast-atom bombardment (f.a.b.) in a glycerol matrix containing ammonium chloride. No characteristic fragment ion was found when trehalose was analysed by either method. In contrast, sucrose gave intense chloride-containing fragments, arising by glycosidic cleavage, when analysed by the first method, whereas such cleavage was not detectable by f.a.b.-ammonium chloride analysis. However, the mass-analysed ion kinetic energy (m.i.k.e.) spectra of the (M + Cl)- ions from either trehalose and sucrose, generated under f.a.b.-ammonium chloride conditions, showed glycosidic cleavage reactions in addition to a large loss of HCl. These cleavage reactions might be attributed to SN2-like reactions on the acetal carbon atom and to base-induced eliminations, and they were enhanced by collision-induced dissociations. However, the relative abundance of such glycosidic cleavages from the ionic state would be too weak to explain the presence of the large chloride-containing fragments in the direct exposure spectra of sucrose. Thus, these ions were mainly produced by a thermal cleavage followed by chloride-attachment reactions.     

Application of capillary gas chromatography-mass spectrometry to chemical characterization of radiation-induced base damage of DNA: implications for assessing DNA repair processes

The application of capillary gas chromatography-mass spectrometry (GC-MS) to the chemical characterization of radiation-induced base products of calf thymus DNA is presented. Samples of calf thymus DNA irradiated in N2O-saturated aqueous solution were hydrolyzed with HCOOH, trimethylsilylated, and subjected to GC-MS analysis using a fused-silica capillary column. Hydrolysis conditions suitable for the simultaneous analysis of the radiation-induced products of all four DNA bases in a single run were determined. The trimethylsilyl derivatives of these products had excellent GC properties and easily interpretable mass spectra; an intense molecular ion (M+.) and a characteristic (M-CH3)+ ion were observed. The complementary use of t-butyldimethylsilyl derivatives was also demonstrated. These derivatives provided an intense characteristic (M-57)+ ion, which appeared as either the base peak or the second most intense ion in the spectra. All mass spectra obtained are discussed. Because of the excellent resolving power of capillary GC and the accurate high-sensitivity identification by MS, the capillary GC-MS is suggested as a very suitable technique for identification of altered bases removed from DNA by base excision-repair enzymes such as DNA glycosylases and, thus, as very useful for an understanding of the base excision-repair of DNA.     

Synthesis and spectroscopic analysis of modified bile salts

For the study of hepatic bile acid transport in vivo, a series of modified bile salts were synthesized. The N-cholyl derivatives of L-leucine, L-alanine, D-alanine, beta-alanine, L-proline, and gamma-amino-butyric acid were prepared from cholic acid, ethyl chloroformate and the corresponding amino acid. Structural analysis of products was carried out mainly by electron impact mass spectrometry (20 eV) of the methyl ester/acetate derivatives. In all EI spectra, fragments in the lower mass region included McLafferty rearrangement ions (beta-cleavage) and product ions of gamma-cleavage in the vicinity of the amide linkage. In the upper mass region, fragmentation was characterized by consecutive eliminations of ketene and/or acetic acid from low intensity molecular ions. The purity of the products and their molecular weights were checked by a novel ionization technique in mass spectrometry, fast atom bombardment (FAB) mass spectrometry. FAB spectra were obtained from underivatized bile salts. The spectra were characterized by ions formed by attachment of a proton or an alkali ion to the bile salt to give intense M+H, M+Na, or M+K ions, which then showed little fragmentation.     

Comparison of electron and chemical ionization mass spectrometry of sialic acids

Sialic acids were analyzed as per-O-trimethylsilylated compounds by gas-liquid chromatography/mass spectrometry either on electron or chemical ionization by isobutane. Electron ionization mass spectra of these derivatives are very similar to those of the corresponding methyl esters described earlier whereas chemical ionization mass spectra are characterized in the high mass range by loss of the C-2 and the C-4 substituents from the M + 1 ion. Together with other fragment ions the seven different sialic acids analyzed could be clearly identified.     

Structural analysis of choline phospholipids by fast atom bombardment mass spectrometry and tandem mass spectrometry

The structures of intact choline phospholipids were determined by positive and negative ion mode fast atom bombardment mass spectrometry, tandem mass spectrometry, and B2/E and B/E constant linked scan mass spectrometry. The molecular weight of the choline lipid could be clearly determined by the appearance of [M + H]+ or [M + Na]+ in the positive ion mode and triplet ions, e.g., [M - 15]-, [M - 60]-, and [M - 86]-, in the negative ion mode. The structures of the triplet ions were assigned to [M - CH3]-, [M - HN(CH3)3]-, and [M - CH2 = CHN(CH3)3]-, respectively, by the MS/MS of each triplet ion, and the origin of the triplet ions was found as the matrix-ion adduct to the target molecule by using the B2/E linked scan technique. The polar group could be identified by the existence of ions indicating glycerophosphocholine and its cleavage products and by the presence of the triplet ions in the negative ion mode. Positional determination of the distribution of constituent fatty acyl groups was carried out by comparing the intensity of deacylated ions from positions 1 and 2 in the positive ion mode and of the ions produced by MS/MS of the triplet ions. From the mass number of the [RCOO]- ion which appeared in the negative ion mode, the molecular weight and degree of unsaturation of the fatty acyl group were determined. The position of double bond(s) in the acyl group was determined from the MS/MS of the [RCOO]- ion.     

Fast atom bombardment and tandem mass spectrometry for structure determination of steroid and flavonoid glycosides

The combination of fast atom bombardment (FAB) and tandem mass spectrometry (MS-MS) was tested for its applicability to generate useful structural information for steroid and flavonoid glycosides. The following compounds were investigated: quercetin, myricitrin, apigetrin, fraxin, rutin, neohesperidin, hesperidin, naringin, apiin, cymarin, digoxin, digitoxin, xanthorhamnin, and frangulin. Upon FAB, the sample molecules are desorbed as (M + H)+, (M - H)-, or as (M + Na)+ or (M + K)+. Collisional activation of (M + H)+ or (M - H)- ions in the MS-MS experiment leads to sequential losses of glycoside moieties in a manner which permits the sequence of glycosides to be established. Some glycosides occur as mixtures of homologs. Proper interpretation of the MS-MS or collisional activation decomposition spectra often allows the homology to be located. In addition to the simple and highly selective fragmentations observed in this combined experiment, FAB and MS-MS also remove interference caused by the ubiquitous matrix ions which are desorbed by FAB.     

Complexes of dibenzylsulfoxide with the first series transition metal nitrates

Under carefully controlled experimental conditions, a series of metal nitrate complexes of dibenzylsulfoxide (DBSO) have been synthesized and isolated (for the dipositive Co, Ni, Cu and Zn elements). The compounds were characterized by use of infrared-, ultraviolet-, and visible spectra. Their stoichiometric compositions appeared to be [M- (DBSO)₃(NO₃)₂], where MCo, Ni and Cu; and [M(DBSO)_3.5(NO₃)₂], where MZn. The additional information concerning the nature of bonding and structural geometry was derived from X-ray diffraction analysis, molecular conductivities, molecular weight and magnetic susceptibility measurements. The infrared spectra indicated that, in all cases, coordination occurred through the oxygen atom of the DBSO ligand. Both analyses and spectral studies suggest that the transition metal ions manifest a coordination number of six. The slight decrease in metal ion radius across the first transition metal series does not affect the number of DBSO molecules bonded to the M²⁺ ion, but the only variation in total coordination of cation might be attributed to the DBSO competition with nitrato groups for the coordination site due to the ability of the anion to enter the coordination sphere.     

Structural characterization of gangliosides isolated from mullet milt using electrospray ionization-tandem mass spectrometry.

Electrospray ionization (ESI) coupled with tandem mass spectrometry has been used in conjunction with microwave-mediated saponification, periodate oxidation, and clostridial sialidase hydrolysis to enable detailed structural characterization of gangliosides and their derivatives present in mullet milt. The gangliosides extracted from mullet milt were determined to be GM3, GM3 lactone, GM3 methyl ester, and 9-O-acetyl GM3. For the major ganglioside GM3 and all GM3 derivatives, the ceramide composition was revealed to be C18:1/C16:0. GM3 with a C18:0/C16:0 ceramide was also found as a minor ganglioside. Both the ganglioside intramolecular ester and the ganglioside methyl ester (lacking carboxylic acid groups) showed dominant chloride attachment peaks (M + Cl)- in negative ion ESI-MS in addition to low intensity peaks corresponding to (M-H)-. GM3 and O-acetyl GM3 bearing carboxylic acid functions showed only (M-H)-. In positive ion ESI, GM3 and O-acetyl GM3 revealed (M + 2Na-H)+ peaks in addition to (M + Na)+, indicating free exchange of the carboxylic acid proton with a sodium cation, while the ganglioside intramolecular ester and ganglioside methyl ester with no acidic protons yielded only (M + Na)+. The strategy of employing ESI-MS to detect products of established wet chemical reactions represents a general approach for elucidation of ganglioside structural details.     

On the metal-ion coordinating properties of the 5'-monophosphates of 1, N6-ethenoadenosine (epsilon-AMP), adenosine and uridine. Comparison of the macrochelate formation in the complexes of epsilon-AMP, AMP, ADP and ATP

The concentration dependence of the chemical shifts of the protons H-2, H-8, H-10, H-11, and H-1' of 1,N6-ethenoadenosine 5'-monophosphate (epsilon-AMP2-) has been measured. The results are consistent with the isodesmic model of indefinite noncooperative stacking; the association constant, K = 2.5 +/- 0.3 M-1, is within experimental error identical to the value determined earlier for AMP2-,K = 2.1 +/- 0.4 M-1. The conditions for the potentiometric pH titrations, used to determine the acidity constants of H2(epsilon-AMP), H2(AMP), and H(UMP)- and the stability constants of the metal ion (M2+) complexes of the corresponding nucleoside 5'-monophosphates (NMP), were chosen so that the ligands were present in the monomeric form. The stabilities of Mg(epsilon-AMP) and Mg(AMP) are similar; however, the stabilities of the Mn2+, Cu2+ and Zn2+ complexes of epsilon-AMP2- are much larger (in the case of Cu2+ by a factor of 700) than those of AMP2-. This is due to the much larger metal ion affinity of the epsilon-adenosine moiety compared to that of the parent adenosine residue. As the uridine moiety does not participate in complex formation, the stability constants of M(UMP) have been used to evaluate the extent of macrochelation (i.e. the simultaneous coordination of M2+ to the base moiety and the phosphate group) in the epsilon-AMP and AMP complexes: the concentration of the macrochelated isomer is considerably larger for M(epsilon-AMP) than for M(AMP). A comparison with previous results for the complexes with ADP3- and ATP4- indicates the order, M(AMP)cl less than M(ADP)-cl greater than M(ATP)2-cl for the tendency to form macrochelates (cl). Due to the relatively high affinity of the epsilon-adenosine moiety towards Mn2+, Cu2+ and Zn2+, the phosphate-monoprotonated complexes M(H . epsilon-AMP)+ also become important; the corresponding complexes play only a minor role in the M2+/AMP systems. Intramolecular aromatic-ring stacking occurs in the ternary Cu(2,2'-bipyridyl)(NMP) complexes: about 80% of Cu(Bpy)(AMP) and Cu(Bpy)(epsilon-AMP) exist as the stacked isomer in aqueous solution; for the former system it has been shown in a previous X-ray study that the intramolecular ligand-ligand interaction occurs also in the solid state [Aoki, K. (1978) J. Am. Chem. Soc. 100, 7106]. Overall, the results emphasize that great care should be exercised in drawing conclusions based on studies of metal-ion-containing enzymic systems in which the natural adenine nucleotide cofactors have been replaced by the corresponding 1,N6-etheno derivatives.     

Structural characterization of neutral glycosphingolipids using high-performance liquid chromatography-electrospray ionization mass spectrometry with a repeated high-speed polarity and MS<Superscript>n</Superscript> switching system

Four types of neutral glycosphingolipids (LacCer, Gb₃Cer, Gb₄Cer, and IV³αGalNAc-Gb₄Cer; 10 pmol each) were analyzed using high-performance liquid chromatography (HPLC)-electrospray ionization quadrupole ion trap time-of-flight (ESI-QIT-TOF) mass spectrometry (MS) with a repeated high-speed polarity and MSⁿ switching system. This system can provide six types of mass spectra, including positive and negative ion MS, MS², and MS³ spectra, within 1 s per cycle. Using HPLC with a normal-phase column, information on the molecular weights of major molecular species of four neutral glycosphingolipids was obtained by detecting [M+Na]⁺ in the positive ion mode mass spectra and [M?H]^? in the negative ion mode mass spectra. Sequences of glycosphingolipid oligosaccharide were obtained in the negative ion MS² spectra. In addition, information on the ceramide structures was clearly obtained in the negative ion MS³ mass spectra. GlcCer molecular species were analyzed by HPLC-ESI-QIT-TOF MS with a reversed-phase column using 1 pmole of GlcCer. The structures of the seven molecular species of GlcCer, namely, d18:1-C16:0, d18:1-C18:0, d18:1-C20:0, d18:1-C22:0, d18:1-C23:0, d18:1-C24:1, and d18:1-C24:0, were characterized using positive ion MS and negative ion MS, MS², and MS³. The established HPLC-ESI-QIT-TOF MS with MSⁿ switching and a normal phase column has been successfully applied to the structural characterization of LacCer and Gb₄Cer in a crude mixture prepared from human erythrocytes.