Nanoelectrospray Ionization Research Articles

Miniaturized SPME tips directly coupled to mass spectrometry for targeted determination and untargeted profiling of small samples

The analysis of small sample volumes, such as biofluids, tissues, and cells, has become increasingly popular over the past few years. In this paper, we introduce a sample-preparation tool that is suitable for such analysis, namely, a solid-phase microextraction (SPME) minitip featuring a tip apex (1 mm) that is coated with biocompatible polyacrylonitrile (PAN) and N-vinylpyrrolidone-co-divinylbenzene, also known as HLB particles. HLB particles are suitable for extracting a wide range of compounds with varying polarities, and the proposed SPME minitips were successfully used for the targeted (MS/MS) quantitation of polar, semi-polar, and nonpolar drugs of abuse (DoAs) in 1 µL of blood via offline nanoelectrospray ionization (nESI). Low limits of detection (LOD) and quantification (LOQ) were obtained for most of the targeted analytes (i.e. LODs between 0.5 and 2.5 ng mL−1). In addition, untargeted metabolomic profiling was performed on a single caviar egg (diameter ≤2.9 mm), with analysis being conducted via liquid chromatography high-resolution mass spectrometry (LC/HRMS). Clear chromatographic peaks were obtained despite the small size of the caviar eggs and the dilution introduced by LC. Multidimensional statistical analysis (PCA & PLS-DA) showed that the SPME minitips were able to successfully discriminate between samples, and the compounds that were tentatively identified corresponded with those expected to be found in fish. These findings highlight the importance of using suitable materials for pre-concentrating probe-sampling approaches, as they can provide analysts with sensitive instrumental response, particularly when studying the chemical profiles of small organisms

Coupling Paternò-Büchi Reaction with Surface-Coated Probe Nanoelectrospray Ionization Mass Spectrometry for In Vivo and Microscale Profiling of Lipid C═C Location Isomers in Complex Biological Tissues.

Lipids are important structural components of biological systems, and lipid C═C locations play important roles in their biophysical and biochemical properties. Rapid, in vivo, in situ, and microscale lipidomics investigation (including precise identification of lipid C═C locations and isomers) of biological specimen has great potential for clinical diagnosis, biological studies, and biomarker discovery. Here we report a novel lipidomics methodology by coupling Paternò-Büchi (PB) reaction with surface-coated probe nanoelectrospray ionization mass spectrometry (SCP-nanoESI-MS) for in vivo, in situ, and microscale analysis of lipid species and C═C location isomers in complex biological tissues. The proposed SCP-PB-nanoESI-MS method was performed by application of a biocompatible solid-phase microextraction (SPME) probe for in vivo, in situ, and microscale sampling and extraction of lipids from biological tissues, and then some spray solvent containing PB reagent was applied to desorb lipid species enriched on SPME probe within a nanospray tip. Subsequently, ultraviolet irradiation was employed to initiate PB reaction for unsaturated lipids within the nanospray tip. After that, a high voltage was applied on the SPME probe to induce nanoESI for MS analysis under ambient and open-air conditions, and collision-induced dissociation was performed to the PB reaction product ions for determination of lipid C═C locations and isomers. By using our proposed SCP-BP-nanoESI-MS method, microscale investigation of lipid compositions and C═C location isomers for lipid droplet of Perilla seed and human intestinal tissue were successfully achieved, and in vivo analysis of lipid species and C═C locations for zebrafish was accomplished.

Direct Analysis of Aqueous Solutions and Untreated Biological Samples Using Nanoelectrospray Ionization Mass Spectrometry with Pipette Tip in Series with High-Ohmic Resistor as Ion Source.

Commercially available disposable plastic pipette tip with the inner diameter of ca. 120μm in series with a high-ohmic resistor (10GΩ) was adapted as a low-cost alternative ion source for high-throughput nanoelectrospray mass spectrometry (nESI-MS) analysis of a variety of samples, especially aqueous solutions, without sample pretreatment. The use of high-ohmic resistor enabled the formation of stable electrospray of aqueous solutions at ambient conditions. In addition, corona discharge was avoided even with a high voltage applied. Quantitative analysis of vitamin B in water was successfully conducted by tip-ESI. The results exhibited a good linearity (R ˃0.9983), a low detection limit (0.25ng/mL), and a wide dynamic response range (0.25-1000ng/mL). Our study revealed that tip-ESI not only performed equally well to capillary nESI in terms of flow rate (˂100nL/min), signal sensitivity, and sample consumption, but also offered a number of additional advantages, including better signal duration, tolerance to high analyte concentration (> 100μg/mL) and high ionizing voltage (up to 6kV), and obviation of tip clogging and corona discharge. High compatibility of tip-ESI with various kinds of samples (aqueous, viscous, solid, or bulk biological samples) makes it a promising tool for direct MS analysis.

Direct target and non-target analysis of urban aerosol sample extracts using atmospheric pressure photoionisation high-resolution mass spectrometry

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous atmospheric pollutants of high concern for public health. In the atmosphere they undergo oxidation, mainly through reactions with ·OH and NOx to produce nitro- and oxygenated (oxy-) derivatives. In this study, we developed a new method for the detection of particle-bound PAHs, nitro-PAHs and oxy-PAHs using direct infusion into an atmospheric pressure photoionisation high-resolution mass spectrometer (APPI-HRMS). Method optimisation was done by testing different source temperatures, gas flow rates, mobile phases and dopants. Samples were extracted with methanol, concentrated by evaporation and directly infused in the APPI source after adding toluene as dopant. Acquisition was performed in both polarity modes. The method was applied to target analysis of seasonal PM2.5 samples from an urban background site in Padua (Italy), in the Po Valley, in which a series of PAHs, nitro- and oxy-PAHs were detected. APPI-HRMS was then used for non-target analysis of seasonal PM2.5 samples and results compared with nano-electrospray ionisation (nanoESI) HRMS. The results showed that, when samples were characterised by highly oxidised organic compounds, including S-containing compounds, like in summer samples, APPI did not bring any additional information with respect to nanoESI in negative polarity (nanoESI(-)). Conversely, for winter samples, APPI(-) could detect a series of aromatic and poly-aromatic compounds, mainly oxidised and nitrogenated aromatics, that were not otherwise detected with nanoESI.

The effects of solution additives and gas-phase modifiers on the molecular environment and conformational space of common heme proteins.

The molecular environment is known to impact the secondary and tertiary structures of biomolecules both in solution and in the gas phase, shifting the equilibrium between different conformational and oligomerization states. However, there is a lack of studies monitoring the impacts of solution additives and gas-phase modifiers on biomolecules characterized using ion mobility techniques. The effect of solution additives and gas-phase modifiers on the molecular environment of two common heme proteins, bovine cytochrome c and equine myoglobin, is investigated as a function of the time after desolvation (e.g., 100-500 ms) using nanoelectrospray ionization coupled to trapped ion mobility spectrometry with detection by time-of-flight mass spectrometry. Organic compounds used as additives/modifiers (methanol, acetonitrile, acetone) were either added to the aqueous protein solution before ionization or added to the ion mobility bath gas by nebulization. Changes in the mobility profiles are observed depending on the starting solution composition (i.e., in aqueous solution at neutral pH or in the presence of organic content: methanol, acetone, or acetonitrile) and the protein. In the presence of gas-phase modifiers (i.e., N2 doped with methanol, acetone, or acetonitrile), a shift in the mobility profiles driven by the gas-modifier mass and size and changes in the relative abundances and number of IMS bands are observed. We attribute the observed changes in the mobility profiles in the presence of gas-phase modifiers to a clustering/declustering mechanism by which organic molecules adsorb to the protein ion surface and lower energetic barriers for interconversion between conformational states, thus redefining the free energy landscape and equilibria between conformers. These structural biology experiments open new avenues for manipulation and interrogation of biomolecules in the gas phase with the potential to emulate a large suite of solution conditions, ultimately including conditions that more accurately reflect a variety of intracellular environments.

Rapid determination of isocitrate dehydrogenase mutation status of human gliomas by extraction nanoelectrospray using a miniature mass spectrometer.

Isocitrate dehydrogenase (IDH) I and II mutations in gliomas cause an abnormal accumulation of 2-hydroxyglutarate (2-HG) in these tumor cells. These mutations have potential prognostic value in that knowledge of the mutation status can lead to improved surgical resection. Information on mutation status obtained by immunohistochemistry or genomic analysis is not available during surgery. We report a rapid extraction nanoelectrospray ionization (nESI) method of determining 2-HG. This should allow the determination of IDH mutation status to be performed intraoperatively, within minutes, using a miniature mass spectrometer. This study demonstrates that the combination of tandem mass spectrometry with low-resolution mass spectrometry allows this analysis to be performed with confidence. Graphical Abstract.

Supercharging of Proteins by Salts during Polarity Reversed Nano-Electrospray Ionization.

Supercharging is beneficial in many ways to the analysis of proteins by mass spectrometry (MS). In this work, a novel supercharging method was developed. It made use of our previously developed ionization technique: namely, polarity reversed nanoelectrospray ionization (PR-nESI) for the ionization of proteins. Supercharging of proteins was achieved by just adding 1-10 mM of a salt to the sample, such as sodium chloride (NaCl). The charge state of proteins obtained by our method was significantly higher than that by nano-ESI with 1% (v/v) acetic acid (HAc). Different kinds of salts were investigated. Salts withstrong acid anions were capable of supercharging proteins, including chlorides, bromides, iodides, and nitrates. The signal intensity and signal to noise ratio ( S/ N) of proteins were increased at the same time. Phosphates were also found to have a supercharging effect, due to the fact that phosphoric acid was a medium-strong acid. In comparison, salts with weak acid anions had no supercharging effect, such as carbonates, sulfides, acetates, and formates. The species of the salt anion was critical to the supercharging effect, while the species of the salt cation showed little influence on the supercharging effect. Investigations were made into the mechanism of our method. The supercharging effect was caused by interactions between protein molecules and salt anions, as well as the influence of protons. The present work offered us an alternative way for the supercharging of proteins. The use of common salts for supercharging made the procedure more convenient. The concentration of salts needed for supercharging was much lower than those conventionally used for supercharging reagents. Taking into consideration the fact that many biological samples are buffered with phosphates and chlorides, these samples could be directly supercharged by our method without any additional additives. Furthermore, as many salts are nontoxic and can easily be found in a chemical laboratory, the use of salts for supercharging would be a much more practical and economical choice. In addition, the present work also furthered our understandings about the mechanism of supercharging, as well as electrospray.

Stable and reproducible nano-electrospray ionization of aqueous solutions and untreated biological samples using ion current limitation combined with polarity reversing

Owing to its high chemical sensitivity and low sample consumption, nanoelectrospray ionization mass spectrometry (nESI-MS) is nowadays widely used in various fields such as chemistry, biology, medicine, pharmaceutical industry, clinical assessment and forensic science.

Online supercritical fluid extraction mass spectrometry (SFE-LC-FTMS) for sensitive characterization of soil organic matter.

We report a novel technical approach for subcritical fluid extraction (SFE) for organic matter characterization in complex matrices such as soil. The custom platform combines on-line SFE with micro-solid phase extraction, nano liquid chromatography (LC), electrospray ionization and Fourier transform mass spectrometry (SFE-LC-FTMS). We demonstrated the utility of SFE-LC-FTMS, including results from both Orbitrap and FTICR MS, for analysis of complex mixtures of organic compounds in a solid matrix by characterizing soil organic matter in peat, a high-carbon soil. For example, in a single experiment, >6000 molecular formulas can be assigned based upon FTICR MS data from 1-50 μL of soil samples (roughly 1-50 mg of soil, dependent on soil density), nearly twice that typically obtained from direct infusion liquid solvent extraction (LSE) from an order of magnitude larger volume of the same soil. The detected species consisted predominately of lipid-like, lignin-like and protein-like compounds, based on their O/C and H/C ratios, with predominantly CHO and CHONP molecular compositions. These results clearly demonstrate that SFE has the potential to effectively extract a variety of molecular species and could become an important member of a suite of extraction methods for studying SOM and other natural organic matter. This is especially true when comprehensive coverage, minimal sample volumes, and high sensitivity are required, or when the presence of organic solvent residue in residual soil is problematic. The SFE based extraction protocol could potentially enable spatially resolved characterization of organic matter in soil with a resolution of ∼1 mm3 to facilitate studies probing the spatial heterogeneity of soil.

Electroosmotic extraction coupled to mass spectrometry analysis of metabolites in live cells.

Quantitative mass spectrometry analysis of metabolites at a single-cell level is critical to understanding the cell functionality and heterogeneity. To preserve cell viability after extraction, the extracted volume needs to be precisely controlled at a subpicoliter-to-picoliter level. Recently, we developed a volume-controlled, and highly sensitive approach for live cell analysis at a single-cell level by integrating electroosmotic extraction and nano-electrospray ionization mass spectrometry (nanoESI MS) analysis. Herein, we use outer epidermal cells of Allium cepa as a model system to present the details of our workflow, including detailed descriptions of the experimental setup for live cell analysis, preparation of the extraction nanopipette, establishment of calibration curves, and extraction and quantification of glucose in an individual onion cell. The capability of this procedure for quantitative live cell analysis has been demonstrated by accurate quantification of glucose in Allium cepa. In principle, our approach is applicable to identification and quantification of metabolites in live mammalian cells.

Online desalting and sequential formation of analyte ions for mass spectrometry characterization of untreated biological samples.

The metal salts ubiquitously present in biological samples cause serious ion suppression, capillary clogging and signal fluctuation in ESI/nESI. Herein, a current-limited high voltage polarity reversing approach was applied for the online separation of intrinsic metal ions in biological samples, resulting in the generation of protonated analytes at the nESI tip for mass analysis without interference from salt cations. Stable and durable signals (∼30-60 s) were observed for protonated proteins, peptides and metabolites in complex biological samples, including liquids, solids and viscous samples, even with very high salt concentration (100 mM NaCl), allowing comprehensive tandem MS analysis with on average ca. 5-times higher analyte signal intensities compared to the conventional nESI analysis. Therefore this approach offers improved performance of nESI/ESI for the sensitive molecular analysis of untreated biological samples, opening new possibilities in various disciplines, including biology, medicine, chemistry, life sciences, etc.

Noninteger Root Transformations for Preprocessing Nanoelectrospray Ionization High-Resolution Mass Spectra for the Classification of Cannabis.

Typically, for measurements with a high dynamic range, the range is reduced by using the square root transform. By using noninteger roots coupled with systematic experimental design, improvements to the measurements may be obtained. The effect of using noninteger root transformation was evaluated using high-resolution mass spectrometry (HRMS) combined with nanoelectrospray ionization (Nano-ESI) to differentiate 23 samples of Cannabis. The mass spectra were evaluated and classified using different mass resolving powers and noninteger root transformations. Classification was achieved by super partial least-squares discriminant analysis (sPLS-DA), support vector machine (SVM), and SVM classification tree type entropy (SVMTreeH). The 2.5 root transformation gave the best overall performance at different resolving powers for chemical profiling from a multilevel factorial experimental design using 2 factors and more than 4 levels. Response surface modeling using a cubic polynomial model of the bootstrapped sPLS-DA average prediction accuracies yielded optima at 0.005 for resolving power and 2.3 for the root transformation. Root transformation is an important spectral preprocessing tool for decreasing the dynamic range so that the relative variance of smaller but more important features may be inflated. For the classification of Cannabis using Nano-ESI, the optimal ranges of root and resolution were broad. The chasing-the-optimum method has been introduced for refining the polynomial response surface model.

Real-Time Electrochemical Detection of Pseudomonas aeruginosa Phenazine Metabolites Using Transparent Carbon Ultramicroelectrode Arrays.

Here, we use a recently developed electrochemical sensing platform of transparent carbon ultramicroelectrode arrays (T-CUAs) for the in vitro detection of phenazine metabolites from the opportunistic human pathogen Pseudomonas aeruginosa. Specifically, redox-active metabolites pyocyanin (PYO), 5-methylphenazine-1-carboxylic acid (5-MCA), and 1-hydroxyphenazine (OHPHZ) are produced by P. aeruginosa, which is commonly found in chronic wound infections and in the lungs of cystic fibrosis patients. As highly diffusible chemicals, PYO and other metabolites are extremely toxic to surrounding host cells and other competing microorganisms, thus their detection is of great importance as it could provide insights regarding P. aeruginosa virulence mechanisms. Phenazine metabolites are known to play important roles in cellular functions; however, very little is known about how their concentrations fluctuate and influence cellular behaviors over the course of infection and growth. Herein we report the use of easily assembled, low-cost electrochemical sensors that provide rapid response times, enhanced sensitivity, and high reproducibility. As such, these T-CUAs enable real-time electrochemical monitoring of PYO and another extremely reactive and distinct redox-active phenazine metabolite, 5-methylphenazine-1-carboxylic acid (5-MCA), from a highly virulent laboratory P. aeruginosa strain, PA14. In addition to quantifying phenazine metabolite concentrations, changes in phenazine dynamics are observed in the biosynthetic route for the production of PYO. Our quantitative results, over a 48-h period, show increasing PYO concentrations during the first 21 h of bacterial growth, after which PYO levels plateau and then slightly decrease. Additionally, we explore environmental effects on phenazine dynamics and PYO concentrations in two growth media, tryptic soy broth (TSB) and lysogeny broth (LB). The maximum concentrations of cellular PYO were determined to be 190 ± 5 μM and 150 ± 1 μM in TSB and LB, respectively. Finally, using desorption electrospray ionization (DESI) and nanoelectrospray ionization (nano-ESI) mass spectrometry we confirm the detection and identification of reactive phenazine metabolites.

The plasma peptidome

BackgroundIt may be possible to discover new diagnostic or therapeutic peptides or proteins from blood plasma using LC–ESI–MS/MS to identify, quantify and compare the statistical distributions of peptides cleaved ex vivo from plasma samples from different clinical populations.MethodsA systematic method for the organic fractionation of plasma peptides was applied to identify and quantify the endogenous tryptic peptides from human plasma from multiple institutions by C18 HPLC followed nano electrospray ionization and tandem mass spectrometry (LC–ESI–MS/MS) with a linear quadrupole ion trap. The endogenous tryptic peptides, or tryptic phospho peptides (i.e. without exogenous digestion), were extracted in a mixture of organic solvent and water, dried and collected by preparative C18. The tryptic peptides from 6 institutions with 12 different disease and normal EDTA plasma populations, alongside ice cold controls for pre-analytical variation, were characterized by mass spectrometry. Each patient plasma was precipitated in 90% acetonitrile and the endogenous tryptic peptides extracted by a stepwise gradient of increasing water and then formic acid resulting in 10 sub-fractions. The fractionated peptides were manually collected over preparative C18 and injected for 1508 LC–ESI–MS/MS experiments analyzed in SQL Server R.ResultsPeptides that were cleaved in human plasma by a tryptic activity ex vivo provided convenient and sensitive access to most human proteins in plasma that show differences in the frequency or intensity of proteins observed across populations that may have clinical significance. Combination of step wise organic extraction of 200 μL of plasma with nano electrospray resulted in the confident identification and quantification ~ 14,000 gene symbols by X!TANDEM that is the largest number of blood proteins identified to date and shows that you can monitor the ex vivo proteolysis of most human proteins, including interleukins, from blood. A total of 15,968,550 MS/MS spectra ≥ E4 intensity counts were correlated by the SEQUEST and X!TANDEM algorithms to a federated library of 157,478 protein sequences that were filtered for best charge state (2+ or 3+) and peptide sequence in SQL Server resulting in 1,916,672 distinct best-fit peptide correlations for analysis with the R statistical system. SEQUEST identified some 140,054 protein accessions, or some ~ 26,000 gene symbols, proteins or loci, with at least 5 independent correlations. The X!TANDEM algorithm made at least 5 best fit correlations to more than 14,000 protein gene symbols with p-values and FDR corrected q-values of ~ 0.001 or less. Log10 peptide intensity values showed a Gaussian distribution from E8 to E4 arbitrary counts by quantile plot, and significant variation in average precursor intensity across the disease and controls treatments by ANOVA with means compared by the Tukey–Kramer test. STRING analysis of the top 2000 gene symbols showed a tight association of cellular proteins that were apparently present in the plasma as protein complexes with related cellular components, molecular functions and biological processes.ConclusionsThe random and independent sampling of pre-fractionated blood peptides by LC-ESI-MS/MS with SQL Server-R analysis revealed the largest plasma proteome to date and was a practical method to quantify and compare the frequency or log10 intensity of individual proteins cleaved ex vivo across populations of plasma samples from multiple clinical locations to discover treatment-specific variation using classical statistics suitable for clinical science. It was possible to identify and quantify nearly all human proteins from EDTA plasma and compare the results of thousands of LC–ESI–MS/MS experiments from multiple clinical populations using standard database methods in SQL Server and classical statistical strategies in the R data analysis system.

The plasma peptides of ovarian cancer

BackgroundIt may be possible to discover new diagnostic or therapeutic peptides or proteins from blood plasma by using liquid chromatography and tandem mass spectrometry to identify, quantify and compare the peptides cleaved ex vivo from different clinical populations. The endogenous tryptic peptides of ovarian cancer plasma were compared to breast cancer and female cancer normal controls, other diseases with their matched or normal controls, plus ice cold plasma to control for pre-analytical variation.MethodsThe endogenous tryptic peptides or tryptic phospho peptides (i.e. without exogenous digestion) were analyzed from 200 μl of EDTA plasma. The plasma peptides were extracted by a step gradient of organic/water with differential centrifugation, dried, and collected over C18 for analytical HPLC nano electrospray ionization and tandem mass spectrometry (LC–ESI–MS/MS) with a linear quadrupole ion trap. The endogenous peptides of ovarian cancer were compared to multiple disease and normal samples from different institutions alongside ice cold controls. Peptides were randomly and independently sampled by LC–ESI–MS/MS. Precursor ions from peptides > E4 counts were identified by the SEQUEST and X!TANDEM algorithms, filtered in SQL Server, before testing of frequency counts by Chi Square (χ2), for analysis with the STRING algorithm, and comparison of precursor intensity by ANOVA in the R statistical system with the Tukey-Kramer Honestly Significant Difference (HSD) test.ResultsPeptides and/or phosphopeptides of common plasma proteins such as HPR, HP, HPX, and SERPINA1 showed increased observation frequency and/or precursor intensity in ovarian cancer. Many cellular proteins showed large changes in frequency by Chi Square (χ2 > 60, p < 0.0001) in the ovarian cancer samples such as ZNF91, ZNF254, F13A1, LOC102723511, ZNF253, QSER1, P4HA1, GPC6, LMNB2, PYGB, NBR1, CCNI2, LOC101930455, TRPM5, IGSF1, ITGB1, CHD6, SIRT1, NEFM, SKOR2, SUPT20HL1, PLCE1, CCDC148, CPSF3, MORN3, NMI, XTP11, LOC101927572, SMC5, SEMA6B, LOXL3, SEZ6L2, and DHCR24. The protein gene symbols with large Chi Square values were significantly enriched in proteins that showed a complex set of previously established functional and structural relationships by STRING analysis. Analysis of the frequently observed proteins by ANOVA confirmed increases in mean precursor intensity in ZFN91, TRPM5, SIRT1, CHD6, RIMS1, LOC101930455 (XP_005275896), CCDC37 and GIMAP4 between ovarian cancer versus normal female and other diseases or controls by the Tukey–Kramer HSD test.ConclusionHere we show that separation of endogenous peptides with a step gradient of organic/water and differential centrifugation followed by random and independent sampling by LC–ESI–MS/MS with analysis of peptide frequency and intensity by SQL Server and R revealed significant difference in the ex vivo cleavage of peptides between ovarian cancer and other clinical treatments. There was striking agreement between the proteins discovered from cancer plasma versus previous biomarkers discovered in tumors by genetic or biochemical methods. The results indicate that variation in plasma proteins from ovarian cancer may be directly discovered by LC–ESI–MS/MS that will be a powerful tool for clinical research.

Improvement of electrospray stability in negative ion mode for nano-PGC-LC-MS glycoanalysis via post-column make-up flow.

Analysis of glycans via a porous graphitized carbon liquid chromatography (PGC-LC) coupled with electrospray ionization (tandem) mass spectrometry (ESI-MS(/MS)) is a powerful analytical method in the field of glycomics. Isobaric glycan structures can be identified reliably with the help of PGC-LC separation and subsequent identification by ESI-MS(/MS) in negative ion mode. In an effort to adapt PGC-LC-ESI-MS(/MS) to the nano-scale operation, spray instability along the nano-PGC-LC gradient was repeatedly observed on an LTQ Orbitrap Elite mass spectrometer equipped with a standard nano-electrospray ionization source. A stable electrospray was achieved with the implementation of a post-column make-up flow (PCMF). Thereby, acetonitrile was used to supplement the eluate from the nano-PGC-LC column. The improved spray stability enhanced detection and resolution of glycans during the analysis. This was in particular the case for smaller O-glycans which elute early in the high aqueous content regime of the nano-PGC-LC elution gradient. This study introduces PCMF as an easy-to-use instrumental adaptation to significantly improve spray stability in negative ion mode nano-PGC-LC-ESI-MS(/MS)-based analysis of glycans.

Recent advances in capillary electrophoresis-mass spectrometry: Instrumentation, methodology and applications.

Capillary electrophoresis (CE) offers fast and high-resolution separation of charged analytes from small injection volumes. Coupled to mass spectrometry (MS), it represents a powerful analytical technique providing (exact) mass information and enables molecular characterization based on fragmentation. Although hyphenation of CE and MS is not straightforward, much emphasis has been placed on enabling efficient ionization and user-friendly coupling. Though several interfaces are now commercially available, research on more efficient and robust interfacing with nano-electrospray ionization (ESI), matrix-assisted laser desorption/ionization (MALDI) and inductively coupled plasma mass spectrometry (ICP) continues with considerable results. At the same time, CE-MS has been used in many fields, predominantly for the analysis of proteins, peptides and metabolites. This review belongs to a series of regularly published articles, summarizing 248 articles covering the time between June 2016 and May 2018. Latest developments on hyphenation of CE with MS as well as instrumental developments such as two-dimensional separation systems with MS detection are mentioned. Furthermore, applications of various CE-modes including capillary zone electrophoresis (CZE), nonaqueous capillary electrophoresis (NACE), capillary gel electrophoresis (CGE) and capillary isoelectric focusing (CIEF) coupled to MS in biological, pharmaceutical and environmental research are summarized.

De-O-Acetylation of mucin-derived sialic acids by recombinant NanS-p esterases of Escherichia coli O157:H7 strain EDL933

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 strain EDL933 encodes the single chromosomal 9-O-acetylesterase NanS, and several copies of prophage-encoded 9-O-acetylesterases (NanS-p). These enzymes have recently been shown to cleave 5-N-acetyl-9-O-acetyl neuraminic acid (Neu5,9Ac2) to yield de-O-acetylated Neu5Ac, the latter of which may serve as a carbon and/or nitrogen source.In the current study, we investigated the NanS- and NanS-p-mediated digestion of synthetic O-acetylated neuraminic acids and bovine submaxillary glands mucin (BSM)-derived O-acetylneuraminic acids by high-performance thin-layer chromatography (HPTLC) and nano electrospray ionization mass spectrometry (nanoESI MS). Initial HPTLC analyses showed the expected activity of NanS and NanS-p variants for Neu5,9Ac2. However, all tested enzymes were unable to de-O-acetylate 5-N-acetyl-4-O-acetylneuraminic acid (Neu5,4 Ac2) in our test system. The nanoESI MS analysis of neuraminic acids after treatment of BSM with NanS-p gave evidence that NanS-p variants of EHEC O157:H7 strain EDL933 cleave off O-acetyl groups from mono-, di-, and tri-O-acetylated Neu5Ac and N-glycolylneuraminic acid (Neu5Gc), regardless of the carbon positions C7, C8 or C9 of the acetate esters. This enzyme activity leads to neuraminidase-accessible Neu5Ac and Neu5Gc on mucin glycans.Moreover, we could demonstrate by HPTLC analyses that recombinant Bacteroides thetaiotaomicron sialidase (BTSA-His) was able to cleave Neu5Ac and Neu5,9Ac2 from BSM and that the combination of BTSA-His with both NanS-His and NanS-p-His derivatives enhanced the release of de-O-acetylated core Neu5Ac and Neu5Gc from mammalian mucin O-glycans.Growth experiments with EHEC wildtype strain EDL933, its nanS and nanS/nanS-p1a-p7 mutant and exogenous BTSA-His in BSM demonstrated that the presence of BTSA-His enhanced growth of EDL933 and the nanS deletion mutant but not the nanS/nanS-p1a-p7 mutant.Thus, we hypothesize that the expression of sialic acid O-acetylesterases with a broad specificity could be an advantage in competition with the gut microbiota for nutrients and facilitate EHEC colonization in the human large intestine.

A Protein Decomplexation Strategy in Snake Venom Proteomics.

Snake venoms are complex mixtures of proteins and peptides that play vital roles in the survival of venomous snakes. As with their diverse pharmacological activities, snake venoms can be highly variable, hence the importance of understanding the compositional details of different snake venoms. However, profiling venom protein mixtures is challenging, in particular when dealing with the diversity of protein subtypes and their abundances. Here we described an optimized strategy combining a protein decomplexation method with in-solution trypsin digestion and mass spectrometry of snake venom proteins. The approach involves the integrated use of C18 reverse-phase high-performance liquid chromatography (RP-HPLC), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and nano-electrospray ionization tandem mass spectrometry (nano-ESI-LC-MS/MS).

Improving the Performance of the Mini 2000 Mass Spectrometer with a Triboelectric Nanogenerator Electrospray Ionization Source.

Balancing the contradiction between portability and analytical performances of a miniaturized mass spectrometer is vital to extend its on-site applications. In this study, triboelectric nanogenerator (TENG)-driven ion sources were coupled with our home-built Mini 2000 system and applied to the analyses of different samples. Compared with the conventional direct current (DC) nanoelectrospray ionization (nanoESI) source, the ion intensity of the TENG-nanoESI miniature mass spectrometer was improved by ∼3 times. Moreover, maybe due to the different pathways of ion formation in comparison with DC electrospray, TENG electrospray is shown to reduce the salt suppression effect during ionization. With these figures of merit, the direct detection of reserpine in saliva was demonstrated using the TENG-Mini 2000 system.