Desorption electrospray ionization mass spectrometry for lipid characterization and biological tissue imaging

Desorption electrospray ionization mass spectrometry (DESI-MS) imaging of biological samples allows untargeted analysis and structural characterization of lipids ionized from the near-surface region of a sample under ambient conditions. DESI is a powerful and sensitive MS ionization method for 2D and 3D imaging of lipids from direct and unmodified complex biological samples. This review describes the strengths and limitations of DESI-MS for lipid characterization and imaging together with the technical workflow and a survey of applications. Included are discussions of lipid mapping and biomarker discovery as well as a perspective on the future of DESI imaging.     

Analysis of lipids from crude lung tissue extracts by desorption electrospray ionization mass spectrometry and pattern recognition

A method is described using desorption electrospray ionization (DESI) mass spectrometry (MS) to obtain phospholipid mass spectral profiles from crude lung tissue extracts. The measured DESI mass spectral lipid fingerprints were then analyzed by unsupervised learning principal components analysis (PCA). This combined approach was used to differentiate the effect(s) of two vaccination routes on lipid composition in mouse lungs. Specifically, the two vaccination routes compared were intranasal (i.n.) and intradermal (i.d.) inoculation of the Francisella tularensis live vaccine strain (Ft–LVS). Lung samples of control and LVS-inoculated mice were quickly extracted with a methanol/chloroform solution, and the crude extract was directly analyzed by DESI–MS, with a total turnaround time of less than 10 min/sample. All of the measured DESI mass spectra (in both positive and negative ion mode) were compared via PCA, resulting in clear differentiation of mass spectral profiles of i.n.-inoculated mouse lung tissues from those of i.d.-inoculated and control mouse lung tissues. Lipid biomarkers responsible for sample differentiation were identified via tandem MS (MS/MS) measurements or by comparison with mass spectra of lipid standards. The DESI–MS approach described here provided a practical and rapid means to analyze tissue samples without extensive extractions and solvent changes.     

Simultaneous determination of residual veterinary drugs in bovine, porcine, and chicken muscle using liquid chromatography coupled with electrospray ionization tandem mass spectrometry

A simple and rapid method using liquid chromatography coupled with electrospray ionization tandem mass spectrometry (LC/MS/MS) for the simultaneous determination of 130 veterinary drugs and their metabolites in bovine, porcine, and chicken muscle was developed. The drugs (1 to 10 ng/g, in muscle) were extracted from bovine, porcine, or chicken muscles with acetonitrile-methanol (95:5, v/v), and the extracts were delipidated with n-hexane saturated with acetonitrile. The extracts were evaporated, dissolved with methanol, analyzed by liquid chromatography with gradient elution on a C18 column, and determined by electrospray ionization tandem mass spectrometry. The detection limits ranged from 0.03 to 3 ng/g. The quantitation limits ranged from 0.1 to 10 ng/g. One hundred eleven, 122, and 123 drugs from bovine, porcine, and chicken muscle respectively showed recoveries between 70 and 110%.     

Ambient desorption ionization mass spectrometry (DART, DESI) and its bioanalytical applications

In recent years, ambient desorption ionization techniques for mass spectrometry were introduced. Among them, the most established techniques are Direct Analysis in Real Time (DART) and Desorption Electrospray Ionization (DESI). Therefore, the current review focuses on the bioanalytical applications of ambient desorption ionization techniques by the example of DART and DESI mass spectrometry. The potential and also limitations of both ambient mass spectrometry (MS) techniques in such areas, as identification and quantitation of small molecules, coupling DART-MS and DESI-MS with planar chromatography, protein/peptide analysis, as well as molecular imaging applications, are discussed.     

Ambient molecular imaging by desorption electrospray ionization mass spectrometry

Desorption electrospray ionization (DESI) allows the direct analysis of ordinary objects or pre-processed samples under ambient conditions. Among other applications, DESI is used to identify and record spatial distributions of lipids and drug molecules in biological tissue sections. This technique does not require sample preparation other than production of microtome tissue slices and does not involve the use of ionization matrices. This greatly simplifies the procedure and prevents the redistribution of analytes during matrix deposition. Images are obtained by continuously moving the sample relative to the DESI sprayer and the inlet of the mass spectrometer. The timing of the protocol depends on the size of the surface to be analyzed and on the desired resolution. Analysis of organ tissue slices at 250 microm resolution typically takes between 30 min and 2 h.     

Rapid detection of atrazine and its metabolite in raw urine by extractive electrospray ionization mass spectrometry

Herbicides such as atrazine are widely used in the biosphere. Urine analysis is usually performed to evaluate the toxicological effects associated with atrazine exposure. A simple procedure based on the extractive electrospray ionization mass spectrometry (EESI-MS) method was established to detect atrazine and its metabolites in undiluted raw urine without sample pretreatment. A 4.3 × 10⁻¹⁴ g atrazine in spiked raw urine was detected and identified by EESI/MS/MS/MS. The detection limit was found to be 0.4 fg for atrazine (m/z 174) and 0.2 fg for 2-chloro-4, 6-diamino-S-triazine (DACT) (m/z 129) (S/N = 3) in EESI/MS/MS. A linear dynamic range of 4–5 orders of magnitude (r = 0.996) was determined for both atrazine and DACT. A single sample analysis was completed using tandem EESI-MS/MS within 1 min, providing a practical convenient method for rapid analysis of trace amounts of targeted metabolites present in complex matrices. Thus, tandem EESI-MS is potentially useful for previously discovered biomarker detection in multiple applications such as clinical diagnosis, drug discovery and forensic science.     

Interspecific variation in localization of hypericins and phloroglucinols in the genus Hypericum as revealed by desorption electrospray ionization mass spectrometry imaging

Plants of the genus Hypericum are widely known for their therapeutic properties. The most biologically active compounds of this genus are naphtodianthrones and phloroglucinols. Indirect desorption electrospray ionization mass spectrometry (DESI‐MS) imaging allows visualization and localization of secondary metabolites in different plant tissues. This study is focused on localization of major secondary compounds in the leaves of 17 different in vitro cultured Hypericum species classified in 11 sections. Generally, all identified naphtodianthrones, protohypericin, hypericin, protopseudohypericin and pseudohypericin were co‐localized in the dark glands of eight hypericin producing species at the site of their accumulation. The known phloroglucinols, hyperforin, adhyperforin, hyperfirin and some new phloroglucinols with m/z [M ? H]^? 495 and 569 were localized in the translucent and pale cavities within the leaf in the majority of studied species. The comparison of different Hypericum species revealed an interspecific variation in the distribution of the dark and translucent glands corresponding with the localization of hypericins and phloroglucinols. Moreover, similarities in the localization and composition of the phloroglucinols were observed in the species belonging to the same section. Adding to various quantitative studies focused on the detection of secondary metabolites, this work using indirect DESI‐MSI offers additional valuable information about localization of the above‐mentioned compounds.     

Simultaneous quantification of arachidonic acid metabolites in cultured tumor cells using high-performance liquid chromatography/electrospray ionization tandem mass spectrometry.

A validated method is described for the simultaneous analysis of PGE2, 11-, 12-, and 5-HETEs from cultured cells using HPLC negative electrospray ionization tandem mass spectrometry (LC/MS/MS). This method permits quantification of selected individual arachidonic acid metabolites from cell extracts without derivatization, multiple purification steps, or lengthy separation times required by traditional GC-MS- or HPLC-UV -based methods. Accuracy assessments of values calculated using this method showed deviations from nominal values were < or =15%. An average relative deviation of 7% of mean calculated values was observed for values taken on separate days. The lower limit of detection for all metabolites was 1.3 pg. The method was used to quantify arachidonic acid metabolites present in various cancer cell lines after incubation with arachidonic acid and the selective cyclooxygenase-2 inhibitor celecoxib. Results showed that the presence of celecoxib in lung cancer A549 cells reduced production of both PGE2 and 11-HETE in a concentration-dependent manner.     

Separation and identification of corticosterone metabolites by liquid chromatography–electrospray ionization mass spectrometry

High-performance liquid chromatography coupled to atmospheric pressure ionization–electrospray ionization mass spectrometry (API–ESI–MS) was investigated for the analysis of corticosterone metabolites; their characterization was obtained by combining the separation on Zorbax Eclipse XDB C₁₈ column (eluted with a methanol–water–acetic acid gradient) with identification using positive ion mode API–ESI–MS and selected ion analysis. The applicability of this method was verified by monitoring the activity of steroid converting enzymes (20β-hydroxysteroid dehydrogenase and 11β-hydroxysteroid dehydrogenase) in avian intestines.     

Eliminating the interferences from TRIS buffer and SDS in protein analysis by fused-droplet electrospray ionization mass spectrometry

Multiply charged protein ions were detected from the solutions containing a high concentration of tris(hydroxymethyl) aminomethane buffer (TRIS) and sodium dodecyl sulfate (SDS) using fused-droplet electrospray ionization mass spectrometry (FD-ESI/MS). The sample aerosols were generated at ambient temperature with a pneumatic nebulizer commonly used to produce sample aerosols in an atmospheric pressure chemical ionization (APCI) source. The aerosols were carried by nitrogen gas to the tip of a capillary where charged methanol droplets had been continuously generated by electrospraying an acidic methanol solution. The neutral sample aerosols then fused with the charged methanol droplets and electrospray ionization proceeded from the newly formed fused droplets to generate multiply charged protein ions. Because of its low solubility in methanol, TRIS molecules (concentration as high as 1 M) were efficiently excluded from the newly formed droplets and the protein ion signals were detected and observed in the mass spectra. To remove the interferences from SDS, equal moles of positively charged cetyltrimethylammonium bromide (CTAB) was added into the SDS containing sample solution to form the dodecyl sulfate-cetyltrimethylammonium ion pair (DS-CTA). The DS-CTA ion pair has a low polarity and solubility in methanol and is excluded from the fused droplet. Protein ions were still detected from the solution containing 10(-2) M of SDS.     

Study of the Se-containing metabolomes in Se-rich yeast by size-exclusion-cation-exchange HPLC with the parallel ICP MS and electrospray orbital ion trap detection

Strong cation exchange HPLC with the parallel ICP MS and electrospray hybrid linear ion trap quadrupole orbital trap mass spectrometry (ESI Orbitrap MS) detection was developed for the study of the metabolomic pattern of selenium in selenium-rich yeast. The mobile phase composition (gradient of ammonium formate in 20% methanol) was optimized to obtain separation in conditions guaranteeing the identical ICP MS sensitivity during the entire chromatographic run and the compatibility with electrospray ionization. Twenty seven Se-containing metabolites observed in the HPLC-ICP MS chromatogram were identified by ESI Orbitrap MS based on the Se isotopic pattern, the accurate molecular mass, and the multistage fragmentation patterns. The method allowed for the first time the correlation of the differences observed in HPLC-ICP MS chromatography of water extracts of Se-rich yeast samples from different manufacturers with the identity of the eluted compounds determined by ESI MS.     

Evaluation of electrospray ionization and atmospheric pressure chemical ionization for simultaneous detection of estrone and its metabolites using high-performance liquid chromatography/tandem mass spectrometry

The levels of estrogens and/or their metabolites play important roles in carcinogenesis, reproductive function, and sexual development during perinatal and adolescence periods. The main purpose of this report was to investigate the applicability of high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) with electrospray ionization (ESI) and/or atmospheric pressure chemical ionization (APCI) for simultaneous detection of estrone (E1) and its six metabolites. Both positive and negative ionization modes in ESI and APCI were used to evaluate the signal responses of seven target analytes. Among the seven target analytes, five analytes, E1, 16alpha-hydroxyestrone, 2-methoxyestrone, 4-methoxyestrone, and 2-hydroxyestrone-3-methyl, produced signals with the best signal-to-noise (S/N) ratios in positive APCI-MS/MS mode, while the other two analytes, 2-hydroxyestrone and 4-hydroxyestrone, yielded the best S/N ratios in negative ESI-MS/MS mode. Based on the results of the evaluation, HPLC-APCI-MS/MS with switching between positive and negative modes was recommended for simultaneous detection of E1 and its six metabolites. The proposed analytical scheme was successfully applied in the analysis of cell culture medium of Human liver carcinoma cells treated with varying amounts of 2,3,7,8-tetrachlorodibenzo-p-dioxin.     

High-performance liquid chromatography/electrospray ionization ion-trap mass spectrometry for analysis of oligosaccharides derivatized by reductive amination and N,N-dimethylation

Milk oligosaccharides derivatized by reductive amination with benzylamine followed by N,N-dimethylation (DMBA-oligosaccharides), were analyzed by high-performance liquid chromatography/electrospray ionization ion-trap mass spectrometry (HPLC/ESI-ITMS). Separation of DMBA-oligosaccharides was achieved on a graphitized carbon column eluted with aqueous acetonitrile and the DMBA-oligosaccharides were detected by positive-ion mode ESI-ITMS allowing sample amounts down to approximately 30fmol of single DMBA-oligosaccharides injected on the HPLC column. MS/MS operation of the mass spectrometer resulted in the detection of diagnostic fragments, mainly belonging to the Y-series, allowing differentiation between isomeric milk oligosaccharides. HPLC/ESI-ITMS/MS/MS experiments indicated the migration of fucose residues of the DMBA milk oligosaccharides to the modified reducing end glucose residue during analysis, a migration previously only observed for proton adduct ions.     

Structural determination of N-linked glycans by matrix-assisted laser desorption/ionization and electrospray ionization mass spectrometry

This paper reviews methods for the analysis of N-linked glycans by mass spectrometry with emphasis on studies conducted at the Oxford Glycobiology Institute. Topics covered are the release of glycans from sodium dodecyl sulphate-polyacrylamide gel electrophoresis gels, their purification for analysis by mass spectrometry, methods based on matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization for producing fragment ions, and details of their fragmentation. MALDI mass spectrometry provided a rapid method for profiling neutral N-linked glycans as their [M + Na](+) ions which could be fragmented by collision-induced decomposition to give spectra containing both glycosidic and cross-ring fragments. Electrospray ionization mass spectrometry was more versatile in that it was relatively easy to change the type of ion that was formed and, furthermore, unlike MALDI, electrospray did not cause extensive loss of sialic acids from sialylated glycans. Negative ions formed by addition of anions such as chloride and, particularly, nitrate, to the electrospray solvent were stable and enabled singly charged ions to be obtained from larger glycans than was possible in positive ion mode. Fragmentation of negative ions followed specific pathways that defined structural details of the glycans that were difficult to obtain by classical methods such as exoglycosidase digestion.     

Identification and quantification of mutagenic halogenated cytosines by gas chromatography,fast atom bombardment,and electrospray ionization tandem mass spectrometry

Oxidative modification of nucleic acids has been implicated in carcinogenesis. One potential mechanism involves halogenation by the myeloperoxidase and eosinophil peroxidase systems of phagocytes. In the current studies, three mass spectrometric methods for the in vitro and in vivo analysis of halogenated cytosines and deoxycytidines were compared: gas chromatography-electron ionization-mass spectrometry (GC-EI-MS) with a quadrupole instrument, fast atom bombardment or electrospray ionization (ESI) tandem MS with a four-sector magnetic instrument, and liquid chromatography ESI tandem MS (HPLC-ESI-MS/MS) with an ion-trap instrument. GC-EI-MS with selected ion monitoring of dimethyl-tert-butylsilyl derivatives of nucleobases was the most sensitive method. High-energy collisionally induced dissociation MS/MS analysis with a four-sector magnetic instrument yielded detailed structural information about halogenated nucleoside adducts but required relatively large amounts of material. The most sensitive analysis of intact halogenated deoxycytidine was achieved with extracted ion chromatograms using HPLC-ESI-MS/MS with an ion-trap instrument. Our results indicate that GC-EI-MS is the methodology of choice for ultrasensitive analysis of halogenated cytosines. HPLC-ESI-MS/MS provides greater structural detail for these compounds and may rival GC-EI-MS in sensitivity with more advanced liquid chromatography applications. The mass spectrometric methods we have developed should be useful for evaluating the role of phagocyte-derived oxidants in halogenating nucleobases, nucleosides, and DNA at sites of inflammation.     

Charge state and adduct reduction in electrospray ionization-mass spectrometry using solvent vapor exposure

The benefits of lowering protein ion charge states in electrospray ionization (ESI) have attracted recent interest. We describe a simple approach to decrease protein charge states by exposure of electrospray droplets to neutral solvent vapor such as acetonitrile. The technique allows detection of weak noncovalent complexes, provides preferred charge states for tandem mass spectrometry (MS/MS) dissociation of protein complexes, and has the added benefit of reducing common adducts, such as alkali metals, without the addition of solution additives or the requirement for a secondary spray.     

Characterization of betaines using electrospray MS/MS

Betaines are an important class of naturally occurring compounds that function as compatible solutes or osmoprotectants. Because of the permanent positive charge on the quaternary ammonium moiety, mass spectrometric analysis has been approached by desorption methods, including fast atom bombardment and plasma desorption mass spectrometry. Here we show that electrospray ionization MS gives comparable results to plasma desorption MS for a range of authentic betaine standards and betaines purified from plant extracts by ion exchange chromatography. A distinct advantage of electrospray ionization MS over plasma desorption MS is the capability of obtaining product ion spectra via MS/MS of selected parent ions, and hence structural information to discriminate between ions of identical mass.     

Quantitative measurement of different ceramide species from crude cellular extracts by electrospray ionization tandem mass spectrometry (ESI-MS/MS).

Ceramide (CER) is an important signaling molecule involved in a variety of cellular processes, including differentiation, cell growth, and apoptosis. Currently, different techniques are applied for CER quantitation, some of which are relatively insensitive and/or time consuming. Tandem mass spectrometry with its high selectivity and sensitivity is a very useful technique for detection of low abundant metabolites without prior purification or derivatization. In contrast to existing mass spectrometry methods, the developed electrospray tandem mass spectrometry (ESI-MS/MS) technique is capable of quantifying different CER species from crude cellular lipid extracts. The ESI-MS/MS is performed with a continuous flow injection and the use of an autosampler, resulting in a high throughput capability. The collision-induced fragmentation of CER produced, in addition to others, a characteristic fragment of m/z 264, making a precursor ion scan of 264 well suited for CER quantitation. Quantitation is achieved by use of a constant concentration of a non-naturally occurring internal standard C8-CER, together with a calibration curve established by spiking different concentrations of naturally occurring CER. The calibration curves showed linearity over a wide concentration range and sample volumes equivalent to 10 microg of cell protein corresponding to about 20, 000 fibroblasts were sufficient for CER analysis. Moreover this assay showed a detection limit at the subpicomole level. In summary, this methodology enables accurate and rapid analysis of CER from small samples without prior separation steps, thus providing a useful tool for signal transduction research.     

Specialized natural product analysis and chemophenetics of some Turkish endemic Centaurea L. (Asteraceae) taxa by electrospray ionization mass spectrometry fingerprinting and liquid chromatography-tandem mass spectrometry

Specialized natural product analysis of six Turkish endemic and two narrowly distributed Centaurea L. taxa was performed via electrospray ionization mass spectrometry (ESI-MS) fingerprinting and liquid chromatography-tandem mass spectrometry (LC-MS/MS), which is an effective methodology that is widely used for fast screening of complex natural mixtures such as food extracts, but not has not been used as commonly for plant chemophenetics. This method is preferable when it is aimed to compare a large number of plant extracts for chemophenetic purposes and when it is difficult to provide equally good chromatographic separation in all of the extracts. ESI-MS shows the major compounds in fingerprinting extracts. LC-MS/MS provides identification according to fragmentation with the advantage of MS/MS, and validation can be performed in selected reaction monitoring (SRM) mode with simultaneous precursor and product ion scans. Herein, sixteen flavones, four flavonols, four flavanones, two lignans, three sesquiterpene lactones, and four phenolic acids, a total of thirty three substances, were identified tentatively or unambiguously from the extracts. It was concluded that ESI-MS fingerprinting is a suitable method for plant chemophenetics when coupled and validated with LC-MS/MS. Moreover, it was concluded that sesquiterpene lactones, lignans, and flavonoids are suitable for taxonomic purposes in Centaurea owing to species-specific metabolite profiles.