Coupling of fully automated chip-based electrospray ionization to high-capacity ion trap mass spectrometer for ganglioside analysis

NanoMate robot was coupled to a high-capacity ion trap (HCT) mass spectrometer to create a system merging automatic chip-based electrospray ionization (ESI) infusion, ultrafast ion detection, and multistage sequencing at superior sensitivity. The interface between the NanoMate and HCT mass spectrometer consists of an in-laboratory constructed mounting device that allows adjustment of the robot position with respect to the mass spectrometer inlet. The coupling was optimized for ganglioside (GG) high-throughput analysis in the negative ion mode and was implemented in clinical glycolipidomics for identification and structural characterization of anencephaly-associated species. By NanoMate HCT mass spectrometry (MS), data corroborating significant differences in GG expression in anencephalic versus age-matched normal brain tissue were collected. The feasibility of chip-based nanoESI HCT multistage collision-induced dissociation (CID MSⁿ) for polysialylated GG fragmentation and isomer discrimination was tested on a GT1 (d18:1/18:0) anencephaly-associated structure. MS²-MS⁴ obtained by accumulating scans at variable fragmentation amplitudes gave rise to the first fragmentation patterns from which the presence of GT1b structural isomer could be determined unequivocally without the need for supplementary investigation by any other analytical or biochemical methods.     

Rapid analysis of Listeria monocytogenes cell wall teichoic acid carbohydrates by ESI-MS/MS

We report the application of electrospray ionization (ESI) mass spectrometry for compositional characterization of wall teichoic acids (WTA), a major component of gram-positive bacterial cell walls. Tandem mass spectrometry (ESI-MS/MS) of purified and chemically hydrolyzed monomeric WTA components provided sufficient information to identify WTA monomers and their specific carbohydrate constituents. A lithium matrix was used for ionization of uncharged WTA monomers, and successfully applied to analyze the WTA molecules of four Listeria strains differing in carbohydrate substitution on a conserved polyribitol-phosphate backbone structure. Carbohydrate residues such as N-acetylglucosamine or rhamnose linked to the WTA could directly be identified by ESI-MS/MS, circumventing the need for quantitative analysis by gas chromatography. The presence of a terminal N-acetylglucosamine residue tethered to the ribitol was confirmed using fluorescently labeled wheat-germ agglutinin. In conclusion, the mass spectrometry method described here will greatly facilitate compositional analysis and characterization of teichoic acids and similar macromolecules from diverse bacterial species, and represents a significant advance in the identification of serovar-specific carbohydrates and sugar molecules on bacteria.     

Improvement of chemical analysis of antibiotics: XXIII. Identification of residual tetracyclines in bovine tissues by electrospray high-performance liquid chromatography–tandem mass spectrometry

To reliably identify the residual tetracycline antibiotics (TCs), oxytetracycline (OTC), tetracycline, chlortetracycline (CTC) and doxycycline (DC), in bovine tissues, we have established a confirmation method using electrospray ionization liquid chromatography–tandem mass spectrometry (ESI LC–MS–MS) with daughter ion scan. All TCs gave [M+H−NH₃]⁺ and [M+H−NH₃−H₂O]⁺ as the product ions, except for DC when [M+H]⁺ was selected as the precursor ion. The combination of C₁₈ cartridge clean-up and the present ESI LC–MS–MS method can reliably identify TCs fortified at a concentration of 0.1 ppm in bovine tissues, including liver, kidney and muscle, and has been successfully applied to the identification of residual OTC in bovine liver and residual CTC in bovine muscle samples previously found at concentrations of 0.58 ppm and 0.38 ppm by LC, respectively.     

Improvement of chemical analysis of antibiotics: XXIII. Identification of residual tetracyclines in bovine tissues by electrospray high-performance liquid chromatography–tandem mass spectrometry

To reliably identify the residual tetracycline antibiotics (TCs), oxytetracycline (OTC), tetracycline, chlortetracycline (CTC) and doxycycline (DC), in bovine tissues, we have established a confirmation method using electrospray ionization liquid chromatography–tandem mass spectrometry (ESI LC–MS–MS) with daughter ion scan. All TCs gave [M+H−NH₃]⁺ and [M+H−NH₃−H₂O]⁺ as the product ions, except for DC when [M+H]⁺ was selected as the precursor ion. The combination of C₁₈ cartridge clean-up and the present ESI LC–MS–MS method can reliably identify TCs fortified at a concentration of 0.1 ppm in bovine tissues, including liver, kidney and muscle, and has been successfully applied to the identification of residual OTC in bovine liver and residual CTC in bovine muscle samples previously found at concentrations of 0.58 ppm and 0.38 ppm by LC, respectively.     

Fast atom bombardment mass spectrometric characterization of peptides

Structural characterization of peptides in the range of 500–5000 Da, using fast atom bombardment (FAB) and Cs⁺ ion liquid secondary ion mass spectrometry (SIMS), is reviewed. These include syntheitc peptides Kemptamide (mol wt 1516); GIF-C15 (mol wt 1875), an isolated natural product as an acylated pentapeptide; and polypeptides generated from enzymatic digests of proteins. MS data is shown to reveal molecular weight and sequence information as well as determine disulfide bonds between cysteine residues and glycosylation sites in the case of a glycopeptide. The complementarity of MS technique to classical biochemical methods for peptide characterization is highlighted. The reader is briefly acquainted with two newer ionization techniques namely, electrospray ionization (ESI) and matrix-assisted laser desorption ionization (MALDI). Synthetic chemists and biochemists can refer to the in-depth review articles that are cited throughout this article.     

Determination of 5-Fluorouracil in Plasma with HPLC-Tandem Mass Spectrometry

In this article, we describe a fast and specific method to measure 5FU with HPLC tandem-mass spectrometry. Reversed-phase HPLC was combined with electrospray ionization tandem mass spectrometry and detection was performed by multiple-reaction monitoring. Stable-isotope-labeled 5FU (1,3–¹⁵N₂–5FU) was used as an internal standard. 5FU was measured within a single analytical run of 16 min with a lower limit of detection of 0.05 μM. The intra-assay variation and inter-assay variation of plasma with added 5FU (1 μM, 10 μM, 100 μM) was less then 6%. Recoveries of the added 5FU in plasma were > 97%. Analysis of the 5FU levels in plasma samples from patients with the HPLC tandem mass spectrometry method and a HPLC-UV method yielded comparable results (r² = 0.98). Thus, HPLC with electrospray ionization tandem mass spectrometry allows the rapid analysis of 5FU levels in plasma and could, therefore, be used for therapeutic drug monitoring.     

α-Chymotrypsin catalyses the synthesis of methotrexate oligomers

The enzymatic synthesis of methotrexate (MTX) catalysed by α -chymotrypsin was studied for the first time. The proteolytic enzyme displayed activity for the synthesis of MTX oligomers composed by 6 repeating units (DP_avg = 1.5). For longer oligomers, molecular dynamics simulations confirmed that as the oligomeric chain grows its accommodation in the enzymes’ active site is hindered, which is evidenced by a decrease of the binding energy associated. The full characterization of the oligomers produced was performed by nuclear magnetic resonance (NMR, ¹H and ¹³C), matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF), electrospray ionization (ESI) and differential scanning calorimetry (DSC).     

Qualitative and quantitative determination of major saponins in Paris and Trillium by HPLC-ELSD and HPLC–MS/MS

High-performance liquid chromatographic (HPLC) with evaporative light scattering detection (ELSD) and HPLC with electrospray ionization multistage tandem mass spectrometry (HPLC–ESI-MSⁿ) were used to identify and quantify steroid saponins in Paris and Trillium plants. The content of the known saponins such as Paris I, II, III, V, VI, VII, H, gracillin and protodioscin in Paris and Trillium plants was determined simultaneously using the developed HPLC-ELSD method. Furthermore, other 12 steroid saponins were identified by HPLC–ESI(+/−)-MSⁿ detection. In the end, a developed analytical procedure was proved to be a reliable and rapid method for the quality control of Paris and Trillium plants. In addition, the alternative resources for Paris yunnanensis used as a traditional Chinese medicine were discovered according to the hierarchical clustering analysis of the saponin fraction of these plants.     

Spectral trees as a robust annotation tool in LC–MS based metabolomics

The identification of large series of metabolites detectable by mass spectrometry (MS) in crude extracts is a challenging task. In order to test and apply the so-called multistage mass spectrometry (MS ⁿ ) spectral tree approach as tool in metabolite identification in complex sample extracts, we firstly performed liquid chromatography (LC) with online electrospray ionization (ESI)?CMS ⁿ , using crude extracts from both tomato fruit and Arabidopsis leaf. Secondly, the extracts were automatically fractionated by a NanoMate LC-fraction collector/injection robot (Advion) and selected LC-fractions were subsequently analyzed using nanospray-direct infusion to generate offline in-depth MS ⁿ spectral trees at high mass resolution. Characterization and subsequent annotation of metabolites was achieved by detailed analysis of the MS ⁿ spectral trees, thereby focusing on two major plant secondary metabolite classes: phenolics and glucosinolates. Following this approach, we were able to discriminate all selected flavonoid glycosides, based on their unique MS ⁿ fragmentation patterns in either negative or positive ionization mode. As a proof of principle, we report here 127 annotated metabolites in the tomato and Arabidopsis extracts, including 21 novel metabolites. Our results indicate that online LC?CMS ⁿ fragmentation in combination with databases of in-depth spectral trees generated offline can provide a fast and reliable characterization and annotation of metabolites present in complex crude extracts such as those from plants.     

Simultaneous quantification of free nucleotides in complex biological samples using ion pair reversed phase liquid chromatography isotope dilution tandem mass spectrometry

A new sensitive and accurate analytical method has been developed for quantification of intracellular nucleotides in complex biological samples from cultured cells of different microorganisms such as Saccharomyces cerevisiae, Escherichia coli, and Penicillium chrysogenum. This method is based on ion pair reversed phase liquid chromatography electrospray ionization isotope dilution tandem mass spectrometry (IP-LC-ESI-ID-MS/MS. A good separation and low detection limits were observed for these compounds using dibutylamine as volatile ion pair reagent in the mobile phase of the LC. Uniformly ¹³C-labeled isotopes of nucleotides were used as internal standards for both extraction and quantification of intracellular nucleotides. The method was validated by determining the linearity, sensitivity, and repeatability.     

The use of mass spectrometry in genomics

Recent advances in the development of electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) now permit the near routine analysis of oligonucleotides and intact nucleic acids. These developments have led to the use of mass spectrometry (MS) as a detection platform for genomics studies. Among the various uses of mass spectrometry in genomics, applications focused on the characterization of single nucleotide polymorphisms (SNPs) and short tandem repeats (STRs) are particularly well-suited to MALDI or ESI-based analysis. It is predicted that continued developments in methodology and instrumentation will further improve the capabilities of mass spectrometry for nucleic acid analysis.     

Matrix Assisted Ionization Vacuum (MAIV), a New Ionization Method for Biological Materials Analysis Using Mass Spectrometry

The introduction of electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) for the mass spectrometric analysis of peptides and proteins had a dramatic impact on biological science. We now report that a wide variety of compounds, including peptides, proteins, and protein complexes, are transported directly from a solid-state small molecule matrix to gas-phase ions when placed into the vacuum of a mass spectrometer without the use of high voltage, a laser, or added heat. This ionization process produces ions having charge states similar to ESI, making the method applicable for high performance mass spectrometers designed for atmospheric pressure ionization. We demonstrate highly sensitive ionization using intermediate pressure MALDI and modified ESI sources. This matrix and vacuum assisted soft ionization method is suitable for the direct surface analysis of biological materials, including tissue, via mass spectrometry.The conversion of large and nonvolatile compounds such as proteins into gas-phase ions is of immense fundamental and practical importance. The 2002 Nobel Prize in Chemistry was awarded for the accomplishment of this conversion via electrospray ionization (ESI)¹ (1) and matrix-assisted laser desorption/ionization (MALDI) (2) interfaced with mass spectrometry (MS) to obtain the molecular weights of proteins with high accuracy. These methods employ high voltage or a laser to form gaseous analyte ions from a wide variety of compounds in solution or a solid matrix, respectively.MALDI interfaced with a time-of-flight (TOF) mass spectrometer produces gas-phase analyte ions in vacuum and is the method of choice for the molecular imaging of biological surfaces. Ionization in vacuum provides excellent ion transmission (3), as well as good spatial resolution achieved using a focused laser beam. However, the analysis of protein complexes is very challenging with MALDI, requiring strategies such as first-shot phenomena (4) and chemical crosslinking (5). The necessity of a laser also makes MALDI less soft than ESI and produces background ions, which can hinder the analysis of small molecules (6, 7). MALDI is also of limited utility on high performance mass-to-charge (m/z) analyzers because of mass range issues related to the formation of singly charged ions, which also produce few fragment ions for structural characterization (8).Multiple charged ions produced directly from solution in ESI bring the m/z ratio within the range of high performance mass spectrometers, allowing the analysis of high-mass compounds. These instruments have advanced features for structural characterization, such as ion mobility spectrometry (IMS) for gas-phase separations (9–11), ultra-high mass resolution and mass accuracy (12–14), and advanced fragmentation such as electron transfer dissociation (ETD) (13, 14). However, ESI is limited for surface characterization, requiring approaches such as desorption-ESI (15) and laser ablation ESI (16), ionization methods that produce multiply charged ions but are not compatible with analyses of larger proteins or fragile complexes.A softer ionization approach is needed in order to observe fragile molecules and molecular complexes in living organisms at low levels directly from tissue and cell cultures, without extensive sample preparation, while retaining spatial information. Ideally, this approach would be compatible with mass spectrometers having advanced capabilities to aid structural characterization directly from surfaces. The new ionization method described here, in which molecules are transferred from solid-phase to gas-phase ions through the simple exposure of a material of interest in a suitable matrix to vacuum, is an advance toward this goal and is of fundamental interest.     

Separation of Organoarsenicals by Means of Zwitterionic Hydrophilic Interaction Chromatography (ZIC®‐HILIC) and Parallel ICP‐MS/ESI‐MS Detection

An analytical scheme was developed for the separation and detection of organoarsenicals using a zwitterionic stationary phase of hydrophilic interaction chromatography (ZIC^®‐HILIC) coupled in parallel to electrospray ionization mass spectrometry (ESI‐MS) and to inductively coupled plasma mass spectroscopy (ICP‐MS). The optimization of separation and detection for organoarsenicals was mainly focused on the influence of the percentage of acetonitrile (MeCN) used as a major component of the mobile phase. Isocratic and gradient elution was applied by varying the MeCN percentage from 78 % to 70 % MeCN and 22 % to 30 % of an aqueous solution of ammonium acetate (125 mM NH₄Ac; pH 8.3) on a ZIC^®‐HILIC column (150 × 2.1 mm id, 3.5 μm), to allow for the separation and successful detection of nine organoarsenicals (i.e., 3‐nitro‐4‐hydroxyphenylarsonic acid (roxarsone, Rox), phenylarsonic acid (PAA), p‐arsanilic acid (p‐ASA), phenylarsine oxide (PAO), dimethylarsinate (DMA), methylarsonate (MMA), arsenobetaine (AsB), arsenocholine (AsC) and trimethylarsine oxide (TMAO)) within 45 min. All analytes were prepared in the mobile phase. The flow rate of the mobile phase, the splitting ratio between ICP‐MS and ESI‐MS detection, and the oxygen addition were adapted to ensure that there appeared a stably burning inductively coupled plasma. Furthermore, the analytical method was evaluated by the identification and quantification of AsB in the reference material DORM‐2 (dogfish muscle) resulting in a 95‐% recovery with respect to the AsB concentration in the extract.     

Quantitative protein analysis using 13C7-labeled iodoacetanilide and d5-labeled N-ethylmaleimide by nano liquid chromatography/nanoelectrospray ionization ion trap mass spectrometry

We have developed a methodology for quantitative analysis and concurrent identification of proteins by the modification of cysteine residues with a combination of iodoacetanilide (IAA, 1) and ¹³C₇-labeled iodoacetanilide (¹³C₇-IAA, 2), or N-ethylmaleimide (NEM, 3) and d₅-labeled N-ethylmaleimide (d₅-NEM, 4), followed by mass spectrometric analysis using nano liquid chromatography/nanoelectrospray ionization ion trap mass spectrometry (nano LC/nano-ESI-IT-MS). The combinations of these stable isotope-labeled and unlabeled modifiers coupled with LC separation and ESI mass spectrometric analysis allow accurate quantitative analysis and identification of proteins, and therefore are expected to be a useful tool for proteomics research.     

Qualitative analysis and simultaneous quantification of phenolic compounds in the aerial parts of Salvia miltiorrhiza by HPLC-DAD and ESI/MS(n)

Introduction – The increasing demands of roots and rhizomes of Salvia miltiorrhiza almost exhausted the wild Salvia sources in China. However, the content and composition of phenolic acids in the aerial parts of the plant and their potential to be used as a substitute has not been explored. Objective – To evaluate the potential of the aerial parts of Salvia miltiorrhiza as new natural sources of phenolic acids. Methodology – HPLC coupled with diode array detection (DAD) and electrospray ionization multistage mass spectrometry (ESI/MSⁿ) has been used for qualitative and quantitative analysis of phenolic compounds. Results – A total of 38 phenolic compounds were identified or tentatively characterized. A quantitative HPLC‐DAD method allowing the simultaneously quantification of six phenolic acids was optimized and validated for linearity, precision, accuracy, and limits of detection and quantification. Calibration curves showed good linear regression (r² > 0.9991) within test ranges; the recoveries ranged between 95.64 and 101.67% and the RSDs were less than 3.01%. Conclusion – The developed methods have been proved to be effective for the identification and quantification of phenolic acids in S. miltiorrhiza. The results obtained suggest that the aerial parts of the plant could be used as an alternative source of sage phenolics. Copyright © 2010 John Wiley & Sons, Ltd.     

Identification of metabolites from liquid chromatography–coulometric array detection profiling: Gas chromatography–mass spectrometry and refractionation provide essential information orthogonal to LC–MS/microNMR

Liquid chromatography–coulometric array detection (LC–EC) is a sensitive, quantitative, and robust metabolomics profiling tool that complements the commonly used mass spectrometry (MS) and nuclear magnetic resonance (NMR)-based approaches. However, LC–EC provides little structural information. We recently demonstrated a workflow for the structural characterization of metabolites detected by LC–EC profiling combined with LC–electrospray ionization (ESI)–MS and microNMR. This methodology is now extended to include (i) gas chromatography (GC)–electron ionization (EI)–MS analysis to fill structural gaps left by LC–ESI–MS and NMR and (ii) secondary fractionation of LC-collected fractions containing multiple coeluting analytes. GC–EI–MS spectra have more informative fragment ions that are reproducible for database searches. Secondary fractionation provides enhanced metabolite characterization by reducing spectral overlap in NMR and ion suppression in LC–ESI–MS. The need for these additional methods in the analysis of the broad chemical classes and concentration ranges found in plasma is illustrated with discussion of four specific examples: (i) characterization of compounds for which one or more of the detectors is insensitive (e.g., positional isomers in LC–MS, the direct detection of carboxylic groups and sulfonic groups in ¹H NMR, or nonvolatile species in GC–MS), (ii) detection of labile compounds, (iii) resolution of closely eluting and/or coeluting compounds, and (iv) the capability to harness structural similarities common in many biologically related, LC–EC-detectable compounds.     

Direct protein detection from biological media through electrospray-assisted laser desorption ionization/mass spectrometry

We report here using a novel technology-electrospray-assisted laser desorption ionization (ELDI)/mass spectrometry-for the rapid and sensitive detection of the major proteins that exist in dried biological fluids (e.g., blood, tears, saliva, serum), bacterial cultures, and tissues (e.g., porcine liver and heart) under ambient conditions. This technique required essentially no sample pretreatment. The proteins in the samples were desorbed using a pulsed nitrogen laser without the assistance of an organic matrix. The desorbed protein molecules were then post-ionized through their fusion into the charged solvent droplets produced from the electrospray of an acidic methanol solution; electrospray ionization (ESI) proceeded from the newly formed droplets to generate the ESI-like protein ions. This new ionization approach combines some of the features of electrospray ionization with those of matrix-assisted laser desorption ionization (MALDI), that is, sampling of a solid surface with spatial resolution, generating ESI-like mass spectra of the desorbed proteins, and operating under ambient conditions.     

Rapid and selective method for norepinephrine in rat urine using reversed-phase ion-pair high-performance liquid chromatography-tandem mass spectrometry

A rugged, high-throughput HPLC–MS–MS-based method, suitable for quantitation of norepinephrine (NE) in urine, has been developed. A rapid, batch-mode procedure utilizes alumina to isolate NE and its deuterated internal standard from urine. After release of NE, using dilute formic acid, samples are analyzed by isocratic reversed-phase ion-pair HPLC, with electrospray ionization (ESI) and MS–MS detection. The ion-pair reagent, heptafluorobutyric acid, is compatible with the ESI interface and permits use of mobile phases with relatively high methanol content, enhancing ESI sensitivity. Furthermore, no significant drop in sensitivity is observed throughout more than 15 h of instrument operation. The selectivity of this approach permitted simplification of the extraction procedure and reduced run times (under 4 min), making single batch-run sizes of more than 200 samples practical. The lower limit of quantitation is 5 ng per 0.5 ml sample, with analytical recoveries of 97–100% and overall method precision of better than 4% relative standard deviation verified up to 500 ng ml⁻¹. This method was initially applied to study the diurnal rhythm in sympathetic nervous system activity of spontaneously hypertensive rats.     

Application of electrospray ionization mass spectrometry in rapid typing of fengycin homologues produced by Bacillus subtilis

AIMS: To rapidly type the fengycin homologues produced by Bacillus subtilis strains with electrospray ionization/collision-induced dissociation (ESI/CID) mass spectrometry. METHODS AND RESULTS: Fengycin homologues produced by Bacillus subtilis JA were analysed. When each homologue was subjected to ESI/CID analysis, ions representing characteristic fragmentations were detected. These ions can help to identify the homologues; even homologues of the same nominal mass can be discriminated by their ESI/CID spectra. Based on the CID results, fengycin homologues can be correctly assigned. CONCLUSIONS AND SIGNIFICANCE OF THIS STUDY: ESI/CID leads to rapid detection and structural characterization of fengycin homologues or lipopeptides with similar properties. It will be very useful in studying the regulatory expression of these peptides.     

Use of isotope differential derivatization for simultaneous determination of thiols and oxidized thiols by liquid chromatography tandem mass spectrometry

Here we report a new isotopic pair of derivatization reagents, ω-bromoacetonylquinolinium bromide (BQB) and d⁷-ω-bromoacetonylquinolinium bromide (d⁷-BQB). BQB and d⁷-BQB both rapidly and selectively reacted with thiols in acidic medium within 3 min with the aid of a microwave. Reduced thiols and total thiols in urine were labeled with BQB and d⁷-BQB, respectively. The BQB- and d⁷-BQB-labeled urine samples were then mixed and separated on a HILIC (hydrophilic interaction chromatography) column followed by electrospray ionization tandem mass spectrometry (ESI–MS/MS) detection. The new strategy, which we have named isotope differential derivatization, allows us to simultaneously determine thiols and oxidized thiols in a single run. Compared with positive mode ESI detection of unlabeled thiols, the positive mode ESI–MS signal intensities of BQB-labeled thiols were found to increase by 10-, 20-, and 40-fold for cysteine (Cys), homocysteine (HCys), and glutathione (GSH), respectively (unlabeled N-acetylcysteine (Nac) is difficult to detect by ESI–MS in positive mode due to its low ionization efficiency). The detection limits calculated at a signal-to-noise ratio of 3 were found to be 8.02, 1.56, 0.833, and 3.27 nmol/L for Cys, HCys, Nac, and GSH, respectively. Recoveries of thiols and disulfides from spiked urine samples were between 80% and 105%. The method was successfully used to determine thiols and oxidized thiols in urine samples of 25 healthy volunteers.