Electrospray ionisation mass spectrometry: principles and clinical applications

This mini-review provides a general understanding of electrospray ionisation mass spectrometry (ESI-MS) which has become an increasingly important technique in the clinical laboratory for structural study or quantitative measurement of metabolites in a complex biological sample. The first part of the review explains the electrospray ionisation process, design of mass spectrometers with separation capability, characteristics of the mass spectrum, and practical considerations in quantitative analysis. The second part then focuses on some clinical applications. The capability of ESI-tandem-MS in measuring bio-molecules sharing similar molecular structures makes it particularly useful in screening for inborn errors of amino acid, fatty acid, purine, pyrimidine metabolism and diagnosis of galactosaemia and peroxisomal disorders. Electrospray ionisation is also efficient in generating cluster ions for structural elucidation of macromolecules. This has fostered a new and improved approach (vs electrophoresis) for identification and quantification of haemoglobin variants. With the understanding of glycohaemoglobin structure, an IFCC reference method for glycohaemoglobin assay has been established using ESI-MS. It represents a significant advancement for the standardisation of HbA1c in diabetic monitoring. With its other applications such as in therapeutic drug monitoring, ESI-MS will continue to exert an important influence in the future development and organisation of the clinical laboratory service.     

Advances in ionisation techniques for mass spectrometry-based omics research

Omics analysis by mass spectrometry (MS) is a vast field, with proteomics, metabolomics and lipidomics dominating recent research by exploiting biological MS ionisation techniques. Traditional MS ionisation techniques such as electrospray ionisation have limitations in analyte-specific sensitivity, modes of sampling and throughput, leading to many researchers investigating new ionisation methods for omics research. In this review, we examine the current landscape of these new ionisation techniques, divided into the three groups of (electro)spray-based, laser-based and other miscellaneous ionisation techniques. Due to the wide range of new developments, this review can only provide a starting point for further reading on each ionisation technique, as each have unique benefits, often for specialised applications, which promise beneficial results for different areas in the omics world.     

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.     

Chemoproteomic approaches to drug target identification and drug profiling

Chemoproteomics represents a new research discipline at the interface of medicinal chemistry, biochemistry, and cell biology focused on studying the molecular mechanisms of action of drugs and other bioactive small molecules. Research strategies frequently combine phenotypic screening with subsequent target identification, and aim at a proteome-wide characterization of drug-induced changes in cellular protein expression and post-translational modifications. In recent years quantitative mass spectrometry has taken center stage in many of these approaches. This review describes experimental strategies in current chemical proteomics research, discusses recent examples of successful applications, and highlights areas in drug discovery where chemical proteomics-based assays using native endogenous proteins are expected to have substantial impact.     

Metabolite signal identification in accurate mass metabolomics data with MZedDB, an interactive m/z annotation tool utilising predicted ionisation behaviour 'rules'

Background  

Metabolomics experiments using Mass Spectrometry (MS) technology measure the mass to charge ratio (m/z) and intensity of ionised molecules in crude extracts of complex biological samples to generate high dimensional metabolite 'fingerprint' or metabolite 'profile' data. High resolution MS instruments perform routinely with a mass accuracy of < 5 ppm (parts per million) thus providing potentially a direct method for signal putative annotation using databases containing metabolite mass information. Most database interfaces support only simple queries with the default assumption that molecules either gain or lose a single proton when ionised. In reality the annotation process is confounded by the fact that many ionisation products will be not only molecular isotopes but also salt/solvent adducts and neutral loss fragments of original metabolites. This report describes an annotation strategy that will allow searching based on all potential ionisation products predicted to form during electrospray ionisation (ESI).     

Positional identification of fluorine in methyl per-O-acetyl-x-deoxy-x-fluoro-alpha-D-hexopyranosides by electron impact and chemical ionisation mass spectrometry.

Fully acetylated methyl x-deoxy-x-fluoro-alpha-D-glucopyranosides have been studied using electron impact and ammonia chemical ionisation mass spectrometry. Mass analysed metastable ion kinetic energy spectroscopy (MIKE), collisional activation (CID), and accelerated voltage scanning have been used to evaluate complete fragmentation schemes. Characteristic differences in the fragmentation of positional isomers were noted on analysis of the spectra, and these make it possible to determine the location of fluorine in the molecules studied. Collisionally activated fragmentation of [M-OCH3]+ ions, produced by electron impact, provides an alternative method for localisation of the fluorine atoms. To the contrary, MIKE and CID spectra of [M + NH4]+ cluster ions produced by chemical ionisation did not afford such structural information.     

Fractionation of grape tannins and analysis by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry

Polymeric tannins, extracted from grape berries (Gamay variety), were fractionated according to their mean degree of polymerisation (mDP) on a styrene-divinylbenzene phase eluted with a gradient of methanol:chloroform. Increasing the percentage of methanol led to the solubilisation of higher molecular weight tannins. The mean mDP of each collected fraction was determined by acid-catalysed degradation in the presence of a nucleophilic reagent. The fractionation method produced a linear gradient of mDP varying between 1.84 and 19.34. The fractions were partially characterised by matrix assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF/MS). The spectra showed a complex mixture of proanthocyanidins and galloylated proanthocyanidins up to 4000 amu.     

Analysis of derivatised steroids by matrix-assisted laser desorption/ionisation and post-source decay mass spectrometry

Neutral steroids are difficult to analyse using desorption ionisation methods coupled with mass spectrometry (MS). However, steroids with an unhindered ketone group can readily be derivatised with the Girard P (GP) reagent to give GP hydrazones. Steroid GP hydrazones contain a quaternary nitrogen atom and are readily desorbed in the matrix-assisted laser desorption/ionisation (MALDI) process, giving an improvement in sensitivity of two orders of magnitude. Steroids without a ketone group, but with a 3beta-hydroxy-Delta5 function, can be readily converted to 3-oxo-Delta4 steroids and subsequently derivatised to GP hydrazones for MALDI analysis. In addition to giving strong [M]+ ions upon MALDI, steroid GP hydrazones give informative post-source decay (PSD) spectra. By using the accurate mass of the precursor-ion measured by MALDI-MS, in combination with the structural information encoded in its PSD spectrum, steroid structures can readily be determined.     

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.     

Capillary liquid chromatography combined with tandem mass spectrometry for the study of neurosteroids and oxysterols in brain

Neurosteroids and neurosterols are found in brain at low levels (ng/g-microg/g) against a high background of cholesterol (mg/g). As such their analysis can be challenging. Traditionally, these molecules have been analysed by gas chromatography (GC)-mass spectrometry (MS), however, the absence of molecular ions in GC-MS spectra, even from derivatised molecules, can make the discovery and identification of novel neurosteroids/sterols difficult. To avoid this scenario, liquid chromatography (LC) combined with desorption ionisation methods are employed. In this review we discuss the application of LC-MS and LC-tandem mass spectrometry (MS/MS) for the identification of neurosteroids/sterols, paying particular attention to the use of low-flow-rate LC to maximise chromatographic and mass spectrometric performance.     

Analysis of glycerophosphoinositol by liquid chromatography-electrospray ionisation tandem mass spectrometry using a beta-cyclodextrin-bonded column

Glycerophosphoinositol (GroPIns) has been demonstrated to have important roles in many intracellular regulatory processes. GroPIns has been analysed for many years by anion-exchange HPLC after radiolabelling of cells in culture, but no method has been developed, to our knowledge, for the direct detection and quantitation of the unlabelled compound in such biological samples. Here is reported a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the direct quantitative analysis of GroPIns that can indeed be applied to cell extracts. Analyses were performed on a beta-cyclodextrin-bonded HPLC column using a binary mobile phase of acetonitrile and 20 mM ammonium formate in water, which allowed direct on-line detection by tandem mass spectrometry in negative electrospray ionisation (ESI) mode. The method was applied to the quantitative analysis of GroPIns in selected rat cell lines after a two-phase acid extraction of cultured cells using external calibration. The potential matrix signal suppression effects were investigated by the parallel quantitation of GroPIns in extracts of selected cultured cell lines with both external calibration and the standard additions method. The accuracy data obtained demonstrated the feasibility of external calibration, so allowing a simpler and less time-consuming approach than that of the standard additions method.     

Characterisation of bacteria by matrix-assisted laser desorption/ionisation and electrospray mass spectrometry

Chemical analysis for the characterisation of micro-organisms is rapidly evolving, after the recent advent of new ionisation methods in mass spectrometry (MS): electrospray (ES) and matrix-assisted laser desorption/ionisation (MALDI). These methods allow quick characterisation of micro-organisms, either directly or after minimum sample preparation. This review provides a brief introduction to ES and MALDI MS and a discussion of micro-organism characterisation capabilities. Some attention is devoted to the analysis of mixtures of proteins, lipids and other compounds, to the combination of polymerase chain reaction technology and MS, and to the analysis of whole bacteria and their lysates. The review of results produced hitherto is concluded with an outlook on future developments.     

Analysis of terfenadine in human plasma using microbore high-performance liquid chromatography–electrospray ionisation mass spectrometry

An assay based on combined microbore high-performance liquid chromatography–positive ion electrospray ionisation mass spectrometry with selected ion recording has been developed for the measurement of the antihistamine drug terfenadine in human plasma. A deuterated analogue of terfenadine was synthesised for use as an internal standard and extraction of terfenadine was carried out on C₁₈ solid phase extraction columns. The limit of detection of terfenadine in plasma is 0.1 ng/ml and the intra-assay coefficient of variation at 1 ng/ml is 10.1%. Plasma concentrations of terfenadine measured in six normal subjects following a 120 mg oral dose are reported.     

Determination of selenium stable isotopes by gas chromatography–mass spectrometry with negative chemical ionisation

A gas chromatography mass spectrometric method using negative chemical ionisation was developed for the determination of stable isotopes of selenium for evaluation of selenium absorption and retention from foods in humans. The method involves an acid digestion to convert all selenium into selenite, which subsequently reacts with 4-nitro-o-phenylene-diamine to form a volatile piazselenole. The piazselenole, after extraction into an organic solvent, was analysed for its isotopic selenium composition by gas chromatography mass spectrometry. Negative chemical ionisation is reported for the first time for the determination of selenium stable isotopes and its analytical characteristics were compared to those of electron impact mass spectrometric ionisation, classically used for the determination of selenium. The negative chemical ionisation technique allowed accurate determination of total selenium by isotope dilution and of selenium isotope ratios in biological samples. The repeatability for total selenium and for stable isotope ratios was good (R.S.D.≤10%) within the range of 50 to 250 ng selenium. The detection limit for the investigated selenium isotopes was approximately 1 pg (signal to noise ratio at 3). The applicability of the developed stable isotope methodology was demonstrated by the determination of the selenium absorption and retention from foods in a pilot study using one human adult.     

Electrospray ionisation–mass spectrometric characterisation of selected anti-psychotic drugs and their detection and determination in human hair samples by liquid chromatography–tandem mass spectrometry

Electrospray ionisation quadrupole ion-trap mass spectrometric (ESI–MS) characterisation of the anti-psychotic drugs chlorpromazine, trifluoperazine, flupenthixol, risperidone and the antidepressant/internal standard trimipramine is presented and possible mechanisms for the observed MSⁿ fragmentation patterns proposed. A validated liquid chromatography (LC)–MS–MS method is then applied to the detection and determination of these drugs in the hair of a patient under clinical treatment for schizophrenia. Chlorpromazine, trifluoperazine and flupenthixol are identified and determined in this hair sample following alkaline degradation of the matrix, solvent extraction and LC–MS–MS using trimipramine as internal standard.     

Determination of the acylcoenzyme A cholesterol acyltransferase inhibitor 447C88 in plasma using gas chromatography-mass spectrometry and liquid chromatography atmospheric pressure chemical ionisation tandem mass spectrometry

Two mass spectrometry-based methods are described for the determination of 447C88 (I), a novel inhibitor of acylcoenzyme A cholesterol acyltransferase (ACAT), in rat, dog and human plasma. The first method uses gas chromatography-mass spectrometry (GC-MS) with electron ionisation and selected-ion monitoring. The method employs solid-phase extraction of I from plasma and requires alkylation of I using iodoethane. The second method uses liquid chromatography-tandem mass spectrometry (LC-MS-MS) with atmospheric-pressure chemical-ionisation and selected-reaction monitoring. The LC-MS-MS method uses a simplified version of the extraction procedure used for GC-MS and does not require derivatisation of I. While both methods provide the necessary limit of quantitation of 0.5 ng/ml in human, dog and rat plasma with the required precision and accuracy, the LC-MS-MS assay offers increased sensitivity, selectivity and speed over the GC-MS assay. This allows a same day turn round of results for in excess of 100 samples, including sample preparation and data acquisition and processing.     

Profiling degradants of paclitaxel using liquid chromatography–mass spectrometry and liquid chromatography–tandem mass spectrometry substructural techniques

A rapid and systematic strategy based on liquid chromatography–mass spectrometry (LC–MS) profiling and liquid chromatography–tandem mass spectrometry (LC–MS–MS) substructural techniques was utilized to elucidate the degradation products of paclitaxel, the active ingredient in Taxol. This strategy integrates, in a single instrumental approach, analytical HPLC, UV detection, full-scan electrospray MS, and MS–MS to rapidly and accurately elucidate structures of impurities and degradants. In these studies, degradants induced by acid, base, peroxide, and light were profiled using LC–MS and LC–MS–MS methodologies resulting in an LC–MS degradant database which includes information on molecular structures, chromatographic behavior, molecular mass, and MS–MS substructural information. The stressing conditions which may cause drug degradation are utilized to validate the analytical monitoring methods and serve as predictive tools for future formulation and packaging studies. Degradation products formed upon exposure to basic conditions included baccatin III, paclitaxel sidechain methyl ester, 10-deacetylpaclitaxel, and 7-epipaclitaxel. Degradation products formed upon exposure to acidic conditions included 10-deacetylpaclitaxel and the oxetane ring opened product. Treatment with hydrogen peroxide produced only 10-deacetylpaclitaxel. Exposure to high intensity light produced a number of degradants. The most abundant photodegradant of paclitaxel corresponded to an isomer which contains a C3–C11 bridge. These methodologies are applicable at any stage of the drug product cycle from discovery through development. This library of paclitaxel degradants provides a foundation for future development work regarding product monitoring, as well as use as a diagnostic tool for new degradation products.     

Proteomic analysis of synaptosomes using isotope-coded affinity tags and mass spectrometry

Synaptosomes are isolated synapses produced by subcellular fractionation of brain tissue. They contain the complete presynaptic terminal, including mitochondria and synaptic vesicles, and portions of the postsynaptic side, including the postsynaptic membrane and the postsynaptic density (PSyD). A proteomic characterisation of synaptosomes isolated from mouse brain was performed employing the isotope-coded affinity tag (ICAT) method and tandem mass spectrometry (MS/MS). After isotopic labelling and tryptic digestion, peptides were fractionated by cation exchange chromatography and cysteine-containing peptides were isolated by affinity chromatography. The peptides were identified by microcapillary liquid chromatography-electrospray ionisation MS/MS (muLC-ESI MS/MS). In two experiments, peptides representing a total of 1131 database entries were identified. They are involved in different presynaptic and postsynaptic functions, including synaptic vesicle exocytosis for neurotransmitter release, vesicle endocytosis for synaptic vesicle recycling, as well as postsynaptic receptors and proteins constituting the PSyD. Moreover, a large number of soluble and membrane-bound molecules serving functions in synaptic signal transduction and metabolism were detected. The results provide an inventory of the synaptic proteome and confirm the suitability of the ICAT method for the assessment of synaptic structure, function and plasticity.     

Near infrared spectroscopy compared to liquid chromatography coupled to mass spectrometry and capillary electrophoresis as a detection tool for peptide reaction monitoring

Peptide interaction is normally monitored by liquid chromatography (LC), liquid chromatography coupled to mass spectrometry (LC-MS), mass spectrometric (MS) methods such as MALDI-TOF/MS or capillary electrophoresis (CE). These analytical techniques need to apply either high pressure or high voltages, which can cause cleavage of newly formed bondages. Therefore, near infrared reflectance spectroscopy (NIRS) is presented as a rapid alternative to monitor the interaction of glutathione and oxytocin, simulating physiological conditions. Thereby, glutathione can act as a nucleophile with oxytocin forming four new conjugates via a disulphide bondage. Liquid chromatography coupled to UV (LC-UV) and mass spectrometry via an electrospray ionisation interface (LC-ESI-MS) resulted in a 82% and a 78% degradation of oxytocin at pH 3 and a 5% and a 7% degradation at pH 6.5. Capillary electrophoresis employing UV-detection (CE-UV) showed a 44% degradation of oxytocin. LC and CE in addition to the NIRS are found to be authentic tools for quantitative analysis. Nevertheless, NIRS proved to be highly suitable for the detection of newly formed conjugates after separating them on a thin layer chromatography (TLC) plate. The recorded fingerprint in the near infrared region allows for a selective distinct qualitative identification of conjugates without the need for expensive instrumentation such as quadrupole or MALDI-TOF mass spectrometers. The performance of the established NIRS method is compared to LC and CE; its advantages are discussed in detail.