Plasmalogen deficiency in early Alzheimer's disease subjects and in animal models: molecular characterization using electrospray ionization mass spectrometry

To explore the hypothesis that alterations in ethanolamine plasmalogen may be directly related to the severity of dementia in Alzheimer's disease (AD), we performed a systematic examination of plasmalogen content in cellular membranes of gray and white matter from different regions of human subjects with a spectrum of AD clinical dementia ratings (CDR) using electrospray ionization mass spectrometry (ESI/MS). The results demonstrate: (1) a dramatic decrease in plasmalogen content (up to 40 mol% of total plasmalogen) in white matter at a very early stage of AD (i.e. CDR 0.5); (2) a correlation of the deficiency in gray matter plasmalogen content with the AD CDR (i.e. approximately 10 mol% of deficiency at CDR 0.5 (very mild dementia) to approximately 30 mol% of deficiency at CDR 3 (severe dementia); (3) an absence of alterations of plasmalogen content and molecular species in cerebellar gray matter at any CDR despite dramatic alterations of plasmalogen content in cerebellar white matter. Alterations of ethanolamine plasmalogen content in two mouse models of AD, APP(V717F) and APPsw, were also examined by ESI/MS. A plasmalogen deficiency was present (up to 10 mol% of total plasmalogen at the age of 18 months) in cerebral cortices, but was absent in cerebella from both animal models. These results suggest plasmalogen deficiency may play an important role in the AD pathogenesis, particularly in the white matter, and suggest that altered plasmalogen content may contribute to neurodegeneration, synapse loss and synaptic dysfunction in AD.     

Substantial sulfatide deficiency and ceramide elevation in very early Alzheimer's disease: potential role in disease pathogenesis

In addition to pathology in the gray matter, there are also abnormalities in the white matter in Alzheimer's disease (AD). Sulfatide species are a class of myelin-specific sphingolipids and are involved in certain diseases of the central nervous system. To assess whether sulfatide content in gray and white matter in human subjects is associated with both the presence of Alzheimer's disease (AD) pathology as well as the stage of dementia, we analyzed the sulfatide content of brain tissue lipid extracts by electrospray ionization mass spectrometry from 22 subjects whose cognitive status at time of death varied from no dementia to very severe dementia. All subjects with dementia had AD pathology. The results demonstrate that: (i) sulfatides were depleted up to 93% in gray matter and up to 58% in white matter from all examined brain regions from AD subjects with very mild dementia, whereas all other major classes of lipid (except plasmalogen) in these subjects were not altered in comparison to those from age-matched subjects with no dementia; (ii) there was no apparent deficiency in the biosynthesis of sulfatides in very mild AD subjects as characterized by the examination of galactocerebroside sulfotransferase activities in post-mortem brain tissues; (iii) the content of ceramides (a class of potential degradation products of sulfatides) was elevated more than three-fold in white matter and peaked at the stage of very mild dementia. The findings demonstrate that a marked decrease in sulfatides is associated with AD pathology even in subjects with very mild dementia and that these changes may be linked with early events in the pathological process of AD.     

Energy-dependent electrospray ionization mass spectrometric studies of mononuclear metal carbonyls

Energy-dependent electrospray ionization mass spectrometry (EDESI-MS) technique has been used for investigating the fragmentation of monomeric metal carbonyl complexes of general formula [M(CO)_x(COOMe)]⁻ (M = Cr, Fe, Mo, and W). The results show that in addition to the loss of CO, formaldehyde loss provides a pathway of fragmentation. Of all complexes investigated, the chromium complex exhibits the cleanest fragmentation. At very high voltages, the metal ions is either associated with a hydride or a methoxide ions.     

Protein sequence and mass spectrometric analyses of tau in the Alzheimer's disease brain.

Tau with unusually slow mobilities in sodium dodecyl sulfate-polyacrylamide gel electrophoresis was purified from the Sarkosyl-insoluble pellet of Alzheimer's disease brain homogenates. Such species of tau (PHF-tau) are considered to construct the framework of the sodium dodecyl sulfate-soluble form of paired helical filaments (PHF). Detailed comparison of peptide maps of PHF-tau and normal tau before and after dephosphorylation pointed to three anomalously eluted peaks which contained abnormally phosphorylated peptides, residues 191-225, 226-240, 260-267, and 386-438, according to the numbering of the longest tau isoform (Goedert, M., Spillantini, M. G., Jakes, R., Rutherford, D., and Crowther, R. A. (1989) Neuron 3, 519-526). Protein sequence and mass spectrometric analyses localized Thr-231 and Ser-235 as the abnormal phosphorylation sites and further indicated that each tau 1 site (residues 191-225) and the most carboxyl-terminal portion of the protein (residues 386-438) carries more than two abnormal phosphates. Ser-262 was also phosphorylated in a fraction of PHF-tau. Modifications other than phosphorylation, removal of the initiator methionine, and N alpha-acetylation at the amino terminus and deamidation at 2 asparaginyl residues were found in PHF-tau, but these modifications were also present in normal tau.     

Determination of huperzine A in formulated products by reversed-phase-liquid chromatography using diode array and electrospray ionization mass spectrometric detection.

A precise and selective high-performance liquid chromatographic (HPLC) method with diode-array detection for quantifying huperzine A in formulated products was developed and validated. A liquid chromatographic-mass spectrometric (LC/MS) procedure was devised to confirm the HPLC method. Huperzine A was dissolved in 1,2-dichloroethane, chromatographed on a YMCBasic C18 column, and detected at 308 nm. A gradient mobile phase of 10 mM ammonium acetate (pH = 3.5)--methanol was used. Identification was based on retention time, UV spectra and mass spectra by comparison with a commercial standard. The UV peak areas were used for quantitation of huperzine A content. The correlation coefficient (R2) of the calibration curve was 1 over the range 0.8-11.6 microg/ml. Overall recovery of huperzine A was 103.9% +/- 1.8 (mean +/- SD). Relative standard deviations for intra- and interday precision were < 2%.     

Low-energy collision-activated dissociation electrospray ionization tandem mass spectrometric analysis of Sinorhizobial succinoglycan monomers

Succinoglycan monomers (M1, M2, and M3) are octasaccharides with acetyl, pyruvyl, and/or succinyl groups as substituents derived from Sinorhizobium meliloti 1021. The dissociation patterns of the octasaccharides caused by low-energy collision-activated dissociation (CAD) were investigated using triple quadrupole tandem mass spectrometry (MS) equipped with an electrospray ionization (ESI) source with increasing collision energy (CE) in negative ion mode. None of the succinoglycan monomers were fragmented at a CE of −25 eV. When the CE was applied to −50 or −70 eV, the loss of the terminal Gal residue and/or the succinyl group of the monomers was observed in the product ion scan mode. Interestingly, the acetyl and the pyruvyl groups in the succinoglycan monomers were not lost even when a CE of −70 eV was applied, indicating that the substituents are more stable than the succinyl group in the octasaccharides.     

Detection of potential new biomarkers of atherosclerosis by probe electrospray ionization mass spectrometry

Introduction

Atherosclerotic diseases are the leading cause of death worldwide. Biomarkers of atherosclerosis are required to monitor and prevent disease progression. While mass spectrometry is a promising technique to search for such biomarkers, its clinical application is hampered by the laborious processes for sample preparation and analysis.

Methods

We developed a rapid method to detect plasma metabolites by probe electrospray ionization mass spectrometry (PESI-MS), which employs an ambient ionization technique enabling atmospheric pressure rapid mass spectrometry. To create an automatic diagnosis system of atherosclerotic disorders, we applied machine learning techniques to the obtained spectra.

Results

Using our system, we successfully discriminated between rabbits with and without dyslipidemia. The causes of dyslipidemia (genetic lipoprotein receptor deficiency or dietary cholesterol overload) were also distinguishable by this method. Furthermore, after induction of atherosclerosis in rabbits with a cholesterol-rich diet, we were able to detect dynamic changes in plasma metabolites. The major metabolites detected by PESI-MS included cholesterol sulfate and a phospholipid (PE18:0/20:4), which are promising new biomarkers of atherosclerosis.

Conclusion

We developed a remarkably fast and easy method to detect potential new biomarkers of atherosclerosis in plasma using PESI-MS.

     

Liquid chromatography–electrospray ionization mass spectrometric analysis of corticosterone in rat plasma using selected ion monitoring

A simple and fast yet highly sensitive and specific method based on HPLC coupled to electrospray ionization mass spectrometry has been developed for the quantitation of corticosterone in rat plasma. After extraction of rat plasma (100 μl) with diethyl ether using 5-pregnen-3β-ol-20-one-16α-carbonitrile (Sigma) as internal standard, HPLC was performed on a short C₈ column (Zorbax-Eclipse, 50×4.6 mm I.D.) using a steep methanol–water gradient (methanol 54% to 90% in 6 min). Detection was performed on a single quadruple mass spectrometer in selected ion monitoring mode (m/z 369 for corticosterone and 364 for the internal standard). The detection limit of the assay was 9 fmol (3 pg) of corticosterone on column. In vitro data were subjected to curve fitting (cubic, r²=0.9999). Recovery of corticosterone after extraction ranged from 81 to 93%. The relative standard deviations for intra- and inter-assay precision ranged from 0.8 to 3.6% and 5.2 to 12.9%, respectively. Corticosterone did not undergo any appreciable degradation when stored in plasma at −20°C for 2 months. The assay is routinely used in our laboratory to examine corticosterone levels as a marker of stress in rats and may also be used for the determination of 18-hydroxy-11-deoxycorticosterone.     

Mass distributions of a macromolecular assembly based on electrospray ionization mass spectrometric masses of the constituent subunits

Macromolecular assemblies containing multiple protein subunits and having masses in the megadalton (MDa) range are involved in most of the functions of a living cell. Because of variation in the number and masses of subunits, macromolecular assemblies do not have a unique mass, but rather a mass distribution. The giant extracelular erythrocruorins (Ers), ∼ 3.5 MDa, comprized of at least 180 polypeptide chains, are one of the best characterized assemblies. Three-dimensional reconstructions from cryoelectron microscopic images show them to be hexagonal bilayer complexes of 12 subassemblies, each comprised of 12 globin chains, anchored to a subassembly of 36 nonglobin linker chains. We have calculated the most probable mass distributions forLumbricus andRiftia assemblies and their globin and linker subassemblies, based on theLumbricus Er stoichiometry and using accurate subunit masses obtained by electrospray ionization mass spectrometry. The expected masses ofLumbricus andRiftia Ers are 3.517 MDa and 3.284 MDa, respectively, with a possible variation of ∼ 9% due to the breadth of the mass distributions. TheLumbricus Er mass is in astonishingly good agreement with the mean of 23 known masses, 3.524 ± 0.481 MDa.     

Targeted metabolomic analysis of Escherichia coli by desorption electrospray ionization and extractive electrospray ionization mass spectrometry

Desorption electrospray ionization (DESI) was utilized to monitor the presence of targeted central carbon metabolites within bacterial cell extracts and the quench supernatant of Escherichia coli. The targeted metabolites were identified through tandem mass spectrometry (MS/MS) product ion scans using collision-induced dissociation in the negative ion mode. Picogram detection limits were achieved for a majority of the metabolites during MS/MS analysis of standard metabolite solutions. In a [U-(13)C]glucose pulse experiment, where uniformly labeled glucose was fed to E. coli, the corresponding fragment ions from labeled metabolites in extracts were generally observed. There was evidence of matrix effects including moderate suppression by other metabolites within the spectra of the labeled and unlabeled extracts. To improve the specificity and sensitivity of detection, optimized in situ ambient chemical reactions using DESI and extractive electrospray ionization (EESI) were carried out for targeted compounds. This study provides the first indication of the potential to perform in situ targeted metabolomics of a bacterial sample via ambient ionization mass spectrometry.     

Kinetic analysis of NodST sulfotransferase using an electrospray ionization mass spectrometry assay

A novel and efficient enzyme kinetics assay using electrospray ionization mass spectrometry was developed and applied to the bacterial carbohydrate sulfotransferase (NodST). NodST catalyzes the sulfuryl group transfer from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to chitobiose, generating 3'-phosphoadenosine 5'-phosphate (PAP) and chitobiose-6-OSO(3)(-) as products. Traditional spectrophotometric assays are not applicable to the NodST system since no shift in absorption accompanies sulfuryl group transfer. Alternative assays have employed thin-layer chromatography, but this procedure is time-consuming and requires radioactive materials. The ESI-MS assay presented herein requires no chromophoric substrate or product, and the analysis time is very short. The ESI-MS assay is used to determine NodST kinetic parameters, including K(M), V(max), and K(i) (for PAP). In addition, the mode of inhibition for PAP was rapidly determined. The results were in excellent agreement with those obtained from previous assays, verifying the accuracy and reliability of the ESI-MS assay. This unique technique is currently being used to investigate the enzymatic mechanism of NodST and to identify sulfotransferase inhibitors.     

Formation and study of single metal ion-phospholipid complexes in biphasic electrospray ionization mass spectrometry

Single metal ion-phospholipid complexes are observed in biphasic electrospray ionization mass spectrometry (BESI-MS) using a dual-channel microsprayer. Such a microsprayer makes it possible to put into contact two immiscible liquids within the Taylor cone. Thus, L-α-dipalmitoyl phosphatidylcholine (DPPC) dissolved in 1,2-dichloroethane (DCE) reacts with aqueous metal cations (M = Na(+), K(+), Ca(2+), Cu(2+), La(3+)) yielding the formation of [M-DPPC(n)](z+) complexes. The number of phospholipid molecules ranges from 1 to 4 for monovalent ions, to 8 for divalent and to more than 10 for trivalent ions respectively. The large number of ligands observed involves the formation of solvent free single ion-phospholipid complexes.     

Development and validation of a liquid chromatographic/electrospray ionization mass spectrometric method for the determination of salidroside in rat plasma: application to the pharmacokinetics study

A sensitive and specific liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) method has been developed and validated for the identification and quantification of salidroside, a major active constituent from Rhodiola rosea L., in rat plasma using helicid as an internal standard. The method involves a simple single-step liquid-liquid extraction with n-butanol. The analytes were separated by isocratic gradient elution on a Shim-pack ODS (4.6 microm, 250 mmx2.0 mm i.d.) column and analyzed in selected ion monitoring (SIM) mode with a negative electrospray ionization (ESI) interface using the respective [M+Cl]- ions, m/z 335 for salidroside, m/z 319 for internal standard. The method was validated over the concentration range of 5-2000 ng/mL for salidroside. Within- and between-batch precision (R.S.D.%) were all within 6% and accuracy ranged from 96 to 112%. The lower limits of quantification was 5 ng/mL. The extraction recovery was on average 86.6% for salidroside. The validated method was used to study the pharmacokinetic profile of salidroside in rat plasma after intravenous and oral administration of salidroside. The bioavailability of salidroside in rats is 32.1%.     

π-Bonded alkene and arene complexes of silver(I): an electrospray mass spectrometric study

Electrospray mass spectra have been observed for a number of alkene and arene complexes of Ag(I) formed by the interaction of AgNO₃ and the organometallic ligand in water/methanol solution. The ES mass spectra show that almost all the alkene and arene ligands in stoichiometric excess form labile 1:2 cationic complexes with Ag(I) which are easily decomposed by collisional activation to the 1:1 species. However, with a deficiency of organic ligand polymeric species are observed. The cation [Ag(cod)₂]⁺ (cod=1,5-cyclooctadiene) was reacted with a variety of other potential ligands, such as PPh₃, AsPh₃, PhSCH₂SPh etc. In most cases, mixed complexes [Ag(cod)(ligand)]⁺ were observed, and excess ligand usually produced [Ag(ligand)₂]⁺.     

Negative electrospray ionization mass spectrometry of synthetic and chemically modified oligonucleotides.

We report here on the analysis of synthetic oligonucleotides by electrospray ionization mass spectrometry (ESI-MS). After intensive removal of salt ions (especially sodium cations), negative ion mass spectra, allowing mass measurement with an accuracy of 0.01%, were obtained on several oligonucleotides up to 80 nucleotides. In most cases, the resolution was sufficient to observe n-1 and n-2 forms due to internal deletions during automated synthesis, and to identify the missing nucleotides. A 132-mer, whose size is close to the limit of automated chemical synthesis, was also successfully mass measured. A quantitative study showed that ESI-MS can provide quantitative data on oligonucleotides of similar size and structure. The described methodology is used to characterize oligonucleotide analogues such as phosphorothioate oligonucleotides designed for antisense applications. Finally, analyses in the positive ion mode on a trimer TpTpT in the presence of different amine bases were performed and allowed a better understanding of the influence of these bases on the ions formation.     

Accurate and rapid modeling of iron-bleomycin-induced DNA damage using tethered duplex oligonucleotides and electrospray ionization ion trap mass spectrometric analysis

Bleomycin B(2)(BLM) in the presence of iron [Fe(II)] and O(2)catalyzes single-stranded (ss) and double-stranded (ds) cleavage of DNA. Electrospray ionization ion trap mass spectrometry was used to monitor these cleavage processes. Two duplex oligonucleotides containing an ethylene oxide tether between both strands were used in this investigation, allowing facile monitoring of all ss and ds cleavage events. A sequence for site-specific binding and cleavage by Fe-BLM was incorporated into each analyte. One of these core sequences, GTAC, is a known hot-spot for ds cleavage, while the other sequence, GGCC, is a hot-spot for ss cleavage. Incubation of each oligo-nucleotide under anaerobic conditions with Fe(II)-BLM allowed detection of the non-covalent ternary Fe-BLM/oligonucleotide complex in the gas phase. Cleavage studies were then performed utilizing O(2)-activated Fe(II)-BLM. No work-up or separation steps were required and direct MS and MS/MS analyses of the crude reaction mixtures confirmed sequence-specific Fe-BLM-induced cleavage. Comparison of the cleavage patterns for both oligonucleotides revealed sequence-dependent preferences for ss and ds cleavages in accordance with previously established gel electrophoresis analysis of hairpin oligonucleotides. This novel methodology allowed direct, rapid and accurate determination of cleavage profiles of model duplex oligonucleotides after exposure to activated Fe-BLM.     

Specific changes of sulfatide levels in individuals with pre‐clinical Alzheimer's disease: an early event in disease pathogenesis

To explore the hypothesis that alterations in cellular membrane lipids are present at the stage of pre‐clinical Alzheimer's disease (AD) (i.e., cognitively normal at death, but with AD neuropathology), we performed targeted shotgun lipidomics of lipid extracts from post‐mortem brains of subjects with pre‐clinical AD. We found sulfatide levels were significantly lower in subjects with pre‐clinical AD compared to those without AD neuropathology. We also found that the level of ethanolamine glycerophospholipid was marginally lower at this stage of AD, whereas changes of the ceramide levels were undetectable with the available samples. These results indicate that cellular membrane defects are present at the earliest stages of AD pathogenesis and also suggest that sulfatide loss is among the earliest events of AD development, while alterations in the levels of ethanolamine glycerophospholipid and ceramide occur relatively later in disease.     

Selective interaction of ethidium derivatives with quadruplexes: an equilibrium dialysis and electrospray ionization mass spectrometry analysis

The telomeric G-rich single-stranded DNA can adopt in vitro an intramolecular quadruplex structure, which has been shown to directly inhibit telomerase activity. The reactivation of this enzyme in immortalized and most cancer cells suggests that telomerase is a relevant target in oncology, and telomerase inhibitors have been proposed as new potential anticancer agents. In this paper, we have analyzed the selectivity of four ethidium derivatives and ethidium itself toward different G-quadruplex species, with electrospray mass spectrometry and competitive equilibrium dialysis and evaluated their inhibitory properties against telomerase. A selectivity profile may be obtained through electrospray ionization mass spectrometry (ESI-MS), which is in fair agreement with competitive equilibrium dialysis data. It also provides unambiguous data on the number of binding sites per nucleic acid (maximal number of two ethidium derivatives per quadruplex, in agreement with external stacking). Our experiments also demonstrate that one compound (4) is the most active and selective G-quadruplex ligand within this series and the most selective telomerase inhibitor in a modified TRAP-G4 assay.     

The observation of chaperone-ligand noncovalent complexes with electrospray ionization mass spectrometry.

Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) was applied for the study of noncovalent chaperone SecB-ligand complexes produced in solution and examined in the gas phase with the aid of electrospray ionization (ESI). Since chaperone proteins are believed to recognize and bind only with ligands with nonnative tertiary structure, this work required careful unfolding of the ligand and subsequent reaction with the intact chaperone (the noncovalent tetrameric protein, SecB). A high denaturant concentration was employed to produce nonnative structures of the OppA, and microdialysis of the resulting solutions containing the chaperone-ligand complexes was carried out to rapidly remove the denaturant prior to analysis. Multistage mass spectrometry was essential to the successful study of these complexes since the initial mass spectra indicated extensive adduction that precluded mass measurements, even after microdialysis. However, low energy collisional activation of the ions in the FTICR trap proved useful for adduct removal, and careful control of excitation level preserved the intact complexes of interest, revealing a 1:1 SecB:OppA stoichiometry. To our knowledge, these results present the first direct observation of chaperone-ligand noncovalent complexes and the highest molecular weight heterogeneous noncovalent complex observed to date by mass spectrometry. Furthermore, these results highlight the capabilities of FTICR for the study of such complex systems, and the development of a greater understanding of chaperone interactions in protein export.