Electrospray ionisation-cleavable tandem nucleic acid mass tag-peptide nucleic acid conjugates: synthesis and applications to quantitative genomic analysis using electrospray ionisation-MS/MS

The synthesis and characterization of isotopomer tandem nucleic acid mass tag-peptide nucleic acid (TNT-PNA) conjugates is described along with their use as electrospray ionisation-cleavable (ESI-Cleavable) hybridization probes for the detection and quantification of target DNA sequences by electrospray ionisation tandem mass spectrometry (ESI-MS/MS). ESI-cleavable peptide TNT isotopomers were introduced into PNA oligonucleotide sequences in a total synthesis approach. These conjugates were evaluated as hybridization probes for the detection and quantification of immobilized synthetic target DNAs using ESI-MS/MS. In these experiments, the PNA portion of the conjugate acts as a hybridization probe, whereas the peptide TNT is released in a collision-based process during the ionization of the probe conjugate in the electrospray ion source. The cleaved TNT acts as a uniquely resolvable marker to identify and quantify a unique target DNA sequence. The method should be applicable to a wide variety of assays requiring highly multiplexed, quantitative DNA/RNA analysis, including gene expression monitoring, genetic profiling and the detection of pathogens.     

Electrospray ionisation-cleavable tandem nucleic acid mass tag–peptide nucleic acid conjugates: synthesis and applications to quantitative genomic analysis using electrospray ionisation-MS/MS

The synthesis and characterization of isotopomer tandem nucleic acid mass tag–peptide nucleic acid (TNT–PNA) conjugates is described along with their use as electrospray ionisation-cleavable (ESI-Cleavable) hybridization probes for the detection and quantification of target DNA sequences by electrospray ionisation tandem mass spectrometry (ESI-MS/MS). ESI-cleavable peptide TNT isotopomers were introduced into PNA oligonucleotide sequences in a total synthesis approach. These conjugates were evaluated as hybridization probes for the detection and quantification of immobilized synthetic target DNAs using ESI-MS/MS. In these experiments, the PNA portion of the conjugate acts as a hybridization probe, whereas the peptide TNT is released in a collision-based process during the ionization of the probe conjugate in the electrospray ion source. The cleaved TNT acts as a uniquely resolvable marker to identify and quantify a unique target DNA sequence. The method should be applicable to a wide variety of assays requiring highly multiplexed, quantitative DNA/RNA analysis, including gene expression monitoring, genetic profiling and the detection of pathogens.     

Probing phospholipid dynamics by electrospray ionisation mass spectrometry

Recent advances in electrospray ionisation mass spectrometry (ESI-MS) have greatly facilitated the analysis of phospholipid molecular species in a growing diversity of biological and clinical settings. The combination of ESI-MS and metabolic labelling employing substrates labelled with stable isotopes is especially exciting, permitting studies of phospholipid synthesis and turnover in vivo. This review will first describe the methodology involved and will then detail dynamic lipidomic studies that have applied the stable isotope incorporation approach. Finally, it will summarise the increasing number of studies that have used ESI-MS to characterise structural and signalling phospholipid molecular species in development and disease.     

Recent studies on the electrospray ionisation mass spectrometric behaviour of selected nitrogen-containing drug molecules and its application to drug analysis using liquid chromatography-electrospray ionisation mass spectrometry

This review presents recent studies on the electrospray ionisation mass spectrometry (ESI-MS) of selected N-containing drug molecules, their metabolites, formulation degradation products and process impurities taken from both studies in the author's laboratory and the recent literature using the Web of Knowledge database. Molecules of mass less than 500 Da are chosen according to selected structural classes in which they give ESI signals primarily in the positive ion mode as [M+H]+ ions. The structural classes are drugs with amine-containing side chains, drugs with N-containing saturated ring structures, drugs with N-containing unsaturated ring structures and quaternary ammonium drugs. Details are given on the fragmentations, where available, that these ionic species exhibit in-source and in ion-trap, triple quadrupole and time-of flight mass spectrometers. Fragmentation data, again where available, using electron impact mass spectrometry (EI-MS) is included for comparison. A review of applications for the period 2004-2005, again taken from the Web of Knowledge database, of the technique liquid chromatography-electrospray ionisation mass spectrometry (LC-ESI-MS) to the detection and determination of these N-containing drug molecules in biomatrices, pharmaceutical formulations, etc., is then made. Analytical information on, for example, sample concentration techniques, LC separation conditions, recoveries from biological media, degradation products and limits of detection (LODs) are provided. Comparisons, where available, are also made with rival analytical techniques such as gas liquid chromatography-mass spectrometry (GLC-MS), capillary electrophoresis-electrospray ionisation mass spectrometry (CE-ESI-MS) and stripping voltammetry (SV).     

Gel-phase synthesis of a difficult sequence peptide on 1,4-butanediol dimethacrylate-crosslinked polystyrene support

The synthetic usefulness of the protocol using NMP/DMSO and DIEA for the synthesis of difficult sequence peptides on amphiphilic and flexible 1,4-butanediol dimethacrylate-crosslinked polystyrene (BDDMA-PS) support was demonstrated by synthesizing [DAla¹⁷] analogue of gonadotropin releasing hormone precursor protein fragment (14–36) [hGnRH (14–36)] using Boc chemistry. The swelling capacity of the peptidyl resin was followed as a measure of the aggregation of pendant peptide chains on the support. The progress of chain assembly was monitored by quantitative ninhydrin test and amino acid analysis. The purity of the peptide was checked by reverse phase HPLC and characterized by amino acid analysis and electrospray ionisation mass spectrometry (ESI-MS).     

Characterisation of intact microorganisms using electrospray ionisation mass spectrometry

We report the first application of electrospray ionisation mass spectrometry (ESI-MS) for the reproducible characterisation of strains of intact Gram-negative and Gram-positive bacteria. Electrospray ionisation was performed in both the positive and negative ion modes and the spectra obtained from Escherichia coli and Bacillus cereus were very information rich. Several of the observed negative mass ion fragments from E. coli could be assigned to specific fragmentation from bacterial phospholipids.Cluster analyses of these spectra showed that ESI-MS could be used to discriminate between these microorganisms to below species level. Therefore we conclude that ESI-MS constitutes a powerful approach to the characterisation and speciation of intact microorganisms.     

Gel-phase synthesis of a difficult sequence peptide on 1,4-butanediol dimethacrylate-crosslinked polystyrene support

Summary The synthetic usefulness of the protocol using NMP/DMSO and DIEA for the synthesis of difficult sequence peptides on amphiphilic and flexible 1,4-butanediol dimethacrylate-crosslinked polystyrene (BDDMA-PS) support was demonstrated by synthesizing [DAla¹⁷] analogue of gonadotropin releasing hormone precursor protein fragment (14–36) [hGnRH (14–36)] using Boc chemistry. The swelling capacity of the peptidyl resin was followed as a measure of the aggregation of pendant peptide chains on the support. The progress of chain assembly was monitored by quantitative ninhydrin test and amino acid analysis. The purity of the peptide was checked by reverse phase HPLC and characterized by amino acid analysis and electrospray ionisation mass spectrometry (ESI-MS).     

Liquid chromatographic tandem mass spectrometric determination of five coccidiostats in poultry eggs and feed

A method is described which permits the quantitative detection of the chemical coccidiostats halofuginone, robenidine, diclazuril, nicarbazin and dimetridazole and its main metabolite 2-hydroxydimetridazole in poultry eggs and feed. Sample preparations were kept very simple and are based upon extraction with an organic solvent. Sample extracts were injected into the liquid chromatography tandem mass spectrometry (LC-MS/MS) system on a C18 column and a gradient elution was performed. Dimetridazole-D3 and diclazuril-bis, a structural analogue of diclazuril, were used as internal standards. Detection was performed on a triple quadrupole mass spectrometer in the selected reaction monitoring mode after ionisation in the positive or negative electrospray ionisation mode. Argon was applied as collision gas for collision induced dissociation. Validation of the methods was performed based on Commission Decision 2002/657/EC [Official Journal of the European Communities L221 (2002) 8].     

Flow infusion electrospray ionisation mass spectrometry for high throughput,non-targeted metabolite fingerprinting: a review

Producing a comprehensive overview of the chemical content of biologically-derived material is a major challenge. Apart from ensuring adequate metabolome coverage and issues of instrument dynamic range, mass resolution and sensitivity, there are major technical difficulties associated with data pre-processing and signal identification when attempting large scale, high-throughput experimentation. To address these factors direct infusion or flow infusion electrospray mass spectrometry has been finding utility as a high throughput metabolite fingerprinting tool. With little sample pre-treatment, no chromatography and instrument cycle times of less than 5 min it is feasible to analyse more than 1,000 samples per week. Data pre-processing is limited to aligning extracted mass spectra and mass-intensity matrices are generally ready in a working day for a month’s worth of data mining and hypothesis generation. ESI-MS fingerprinting has remained rather qualitative by nature and as such ion suppression does not generally compromise data information content as originally suggested when the methodology was first introduced. This review will describe how the quality of data has improved through use of nano-flow infusion and mass-windowing approaches, particularly when using high resolution instruments. The increasingly wider availability of robust high accurate mass instruments actually promotes ESI-MS from a merely fingerprinting tool to the ranks of metabolite profiling and combined with MS/MS capabilities of hybrid instruments improved structural information is available concurrently. We summarise current applications in a wide range of fields where ESI-MS fingerprinting has proved to be an excellent tool for “first pass” metabolome analysis of complex biological samples. The final part of the review describes a typical workflow with reference to recently published data to emphasise key aspects of overall experimental design.

     

Flow infusion electrospray ionisation mass spectrometry for high throughput,non-targeted metabolite fingerprinting: a review

Producing a comprehensive overview of the chemical content of biologically-derived material is a major challenge. Apart from ensuring adequate metabolome coverage and issues of instrument dynamic range, mass resolution and sensitivity, there are major technical difficulties associated with data pre-processing and signal identification when attempting large scale, high-throughput experimentation. To address these factors direct infusion or flow infusion electrospray mass spectrometry has been finding utility as a high throughput metabolite fingerprinting tool. With little sample pre-treatment, no chromatography and instrument cycle times of less than 5 min it is feasible to analyse more than 1,000 samples per week. Data pre-processing is limited to aligning extracted mass spectra and mass-intensity matrices are generally ready in a working day for a month’s worth of data mining and hypothesis generation. ESI-MS fingerprinting has remained rather qualitative by nature and as such ion suppression does not generally compromise data information content as originally suggested when the methodology was first introduced. This review will describe how the quality of data has improved through use of nano-flow infusion and mass-windowing approaches, particularly when using high resolution instruments. The increasingly wider availability of robust high accurate mass instruments actually promotes ESI-MS from a merely fingerprinting tool to the ranks of metabolite profiling and combined with MS/MS capabilities of hybrid instruments improved structural information is available concurrently. We summarise current applications in a wide range of fields where ESI-MS fingerprinting has proved to be an excellent tool for “first pass” metabolome analysis of complex biological samples. The final part of the review describes a typical workflow with reference to recently published data to emphasise key aspects of overall experimental design.     

Mass spectrometric investigation of the DNA-binding properties of an anthracycline with two trisaccharide chains

Cosmomycin D (CosD) is an anthracycline that has two trisaccharide chains linked to its ring system. Gel electrophoresis showed that CosD formed stable complexes with plasmid DNA under conditions where daunorubicin (Dn) and doxorubicin (Dx) dissociated to some extent during the experiments. The footprint and stability of CosD complexed with 10- and 16 mer DNA was investigated using several applications of electrospray ionisation mass spectrometry (ESI-MS). ESI-MS binding profiles showed that fewer CosD molecules bound to the sequences than Dn or Dx. In agreement with this, ESI-MS analysis of nuclease digestion products of the complexes showed that CosD protected the DNA to a greater extent than Dn or Dx. In tandem MS experiments, all CosD–DNA complexes were more stable than Dn- and Dx-DNA complexes. These results support that CosD binds more tightly to DNA and exerts a larger footprint than Dn or Dx. ESI-MS investigations of the binding properties of CosD could be carried out rapidly and using only small amounts of sample.     

Stable isotope analysis of dynamic lipidomics

Metabolic pathway flux is a fundamental element of biological activity, which can be quantified using a variety of mass spectrometric techniques to monitor incorporation of stable isotope-labelled substrates into metabolic products. This article contrasts developments in electrospray ionisation mass spectrometry (ESI-MS) for the measurement of lipid metabolism with more established gas chromatography mass spectrometry and isotope ratio mass spectrometry methodologies. ESI-MS combined with diagnostic tandem MS/MS scans permits the sensitive and specific analysis of stable isotope-labelled substrates into intact lipid molecular species without the requirement for lipid hydrolysis and derivatisation. Such dynamic lipidomic methodologies using non-toxic stable isotopes can be readily applied to quantify lipid metabolic fluxes in clinical and metabolic studies in vivo. However, a significant current limitation is the absence of appropriate software to generate kinetic models of substrate incorporation into multiple products in the time domain. Finally, we discuss the future potential of stable isotope-mass spectrometry imaging to quantify the location as well as the extent of lipid synthesis. This article is part of a Special Issue entitled: BBALIP_Lipidomics Opinion Articles edited by Sepp Kohlwein.     

FAB, ESI and MALDI Mass Spectrometric methods in the study of metallo-drugs and their biomolecular interactions

This review surveys the soft ionisation mass spectrometric methods that are most commonly used for the investigation of the mechanism of interaction between metallo-drugs and biomolecules. In the first part of the review, an overview of the applications of transition metal complexes in the therapy of various diseases (arthritis, cancer, diabetes) is given, whereas the second part focuses on the obtained results dealing with various aspects of the interaction between metal complexes and different types of biomolecules. Possibilities and limitations of each mass spectrometric method-namely, fast atom bombardment (FAB), electrospray ionisation (ESI) and matrix-assisted laser desorption and ionisation (MALDI)--are discussed in the third part of this review along with the examples of their application for the analysis of metal complexes, as well as of the products of their interaction with biomolecules.     

A rapid LC-MS method for determination of plasma anion profiles of acidotic patients

In metabolic acidosis, the concentrations of anions associated with intermediary metabolism are increased and can make a significant contribution to the observed acidosis. Here we describe a method for the rapid determination of the plasma ultrafiltrate profile of these anions using liquid chromatography coupled to electrospray ionisation mass spectrometry (LC/ESI-MS). The ultrafiltrate from patients with acidosis resulting from various causes were examined and the results compared to control values. Using the LC/ESI-MS method described, a unique plasma ultrafiltrate anion profile was obtained for each of the groups studied that provides rapid diagnosis of the type of underlying acidosis.     

A rapid and direct method for the determination of active site accessibility in proteins based on ESI-MS and active site titrations

We have developed an electrospray ionisation mass spectrometry (ESI-MS) technique that can be applied to rapidly determine the number of intact active sites in proteins. The methodology relies on inhibiting the protein with an active-site irreversible inhibitor and then using ESI-MS to determine the extent of inhibition. We have applied this methodology to a test system: a serine protease, subtilisin Carlsberg, and monitored the extent of inhibition by phenylmethylsulfonyl fluoride (PMSF), an irreversible serine hydrolase inhibitor as a function of the changes in immobilisation and hydration conditions. Two types of enzyme preparation were investigated, lyophilised enzymes and protein-coated microcrystals (PCMC).     

Negative-ion electrospray ionisation-mass spectrometry (ESI-MS) as a tool for analysing structural heterogeneity in kappa-carrageenan oligosaccharides

Oligosaccharides, enzymically produced from kappa-carrageenan, have been investigated by electrospray ionisation mass spectrometry (ESI-MS). The technique was used without prior derivatisation of the oligosaccharide originally obtained by size-exclusion chromatography (SEC). The structure of the oligosaccharides was mainly 4-sulphated neocarrabiose (A-G4S) with an increasing length ranging from di- to dodecasaccharides. However, in the larger oligosaccharides, structural motifs deviating from the perfect alternating A-G4S structure were detected, i.e. (A2S-G4S). Although resulting in reduced signal intensity, samples to which NaCl was added also gave rise to reliable mass spectra. Desulphation was induced at elevated cone voltages and in acidic or alkaline salt solutions.     

Determination of fumonisins and hydrolyzed fumonisin B1 in microbial culture media by LC/ESI-MS

In order to analyze liquid growth media for fumonisin B₁, B₂ and hydrolyzed fumonisin B1 (HFB1) after incubation with microorganisms for degradation studies, a liquid chromatography/electrospray ionisation mass spectrometry (LC/ESI-MS) method was developed. The use of phytosphingosine hydrochloride (PSH) and T-2-toxin-d3 (T2d3) as internal standard has been tested. The detection limit established with PSH as an internal standard was about 20 ng/mL for FB1 and FB2 and about 50 ng/mL for HFB1. The developed method allows the rapid, simultaneous and quantitative determination of these analytes in microbial culture media without any cleanup. Presented at the 25^th Mykotoxin Workshop in Giessen, Germany May 19–21, 2003     

Determination of nicotine and cotinine in human serum by means of LC/MS

As part of a joint clinical research project to study the effects of nicotine on the brain, a HPLC electrospray ionisation mass spectrometry method with a solid-phase extraction sample preparation was developed for the quantitative determination of nicotine and cotinine in human serum in volunteers. The measured concentrations of nicotine and cotinine were used as control for smoking behaviour. A X-Bridge-column from Waters, and a SSQ 7000 single quadropole mass spectrometer with a TSP liquid chromatographic system were used. The method includes a simple and robust sample preparation and this assay has been shown to be of a sufficient sensitivity for this application. The limits of quantification were 5 and 2 ng/ml for cotinine and nicotine, respectively. A simultaneous study was conducted to measure nicotine receptor availability and the vigilance in the same group of volunteers.     

Characterisation of a potential biomarker of phospholipidosis from amiodarone-treated rats

A novel and relatively simple analytical method for the separation, characterisation and semi-quantitation of phospholipids (PLs) from extracts of complex biological samples has been developed. This methodology allows PL extracts from cells and tissues to be analysed by liquid chromatography (LC) coupled to electrospray ionisation mass spectrometry (ESI-MS). Complex mixtures of PLs were separated on a high-performance liquid chromatography (HPLC) system using 0.5% ammonium hydroxide in methanol/water/hexane/formate mixture with UV detection at 205 nm. Identification and structural characterisation of molecular species were carried out utilising ESI-MS and MS/MS in the negative ion mode.The abnormal accumulation of PLs (phospholipidosis) was induced in male Sprague-Dawley rats by administration of the cationic amphiphilic drug (CAD), amiodarone. Analysis of the PL profile of liver and lung tissues, lymphocytes and serum from treated rats was carried out using this analytical procedure (LC-ESI/MS/MS). Differences in PL profiles between treated and untreated animals were highlighted by principal component analysis (PCA). This led to the selection of a potential metabolic marker of phospholipidosis (PLD) identified as a lyso-bis-phosphatidic acid (LBPA) derivative, also known as bis(monoglycero)phosphate (BMP). This PL was absent in control animals but was present in quantifiable amounts in all samples from amiodarone-treated rats.     

Identification of bifunctional GA and AG intrastrand crosslinks formed between cisplatin and DNA

A combination of enzymatic digestion and electrospray ionisation mass spectrometry (ESI-MS) was used to characterise bifunctional adducts in which cisplatin is bound to GA base sequences in 8mer and 16mer oligonucleotides that do not contain other, higher affinity binding sites. The extent of formation of bifunctional adducts with GA base sequences was significant, but less than that seen with similar oligonucleotides containing either AG or GG sequences.