Dual Emitter Nano-Electrospray Ionization Coupled to Differential Ion Mobility Spectrometry-Mass Spectrometry for Shotgun Lipidomics

Current lipidomics workflows are centered around acquisition of large data sets followed by lengthy data processing. A dual nESI-DIMS-MS platform was developed to perform real-time relative quantification between samples, providing data required for biomarker discovery and validation more quickly than traditional ESI-MS approaches. Nanosprayer activity and DIMS compensation field settings were controlled by a LabVIEW program synced to the accumulation portion of the ion trap scan function, allowing for full integration of the platform with a commercial mass spectrometer. By comparing samples with short electrospray pulses rather than constant electrospray, the DIMS and MS performance is normalized within an experiment, as signals are compared between individual mass spectra (ms time scale) rather than individual experiments (min–hr time scale). The platform was validated with lipid standards and extracts from nitrogen-deprived microalgae. Dual nESI-DIMS requires minimal system modification and is compatible with all traditional ion activation techniques and mass analyzers, making it a versatile improvement to shotgun lipidomics workflows

Distinguishing Linkage Position and Anomeric Configuration of Glucose–Glucose Disaccharides by Water Adduction to Lithiated Molecules

A method was developed to distinguish both the linkage position and the anomericity of all reducing and two nonreducing glucopyransosyl–glucose disaccharides using only electrospray ionization–mass spectrometry/mass spectrometry (ESI–MS/MS). Carbohydrates are well-known to form complexes with metal cations during electrospray ionization. Addition of a lithium salt to a solution containing a disaccharide, M, results in [M + Li]⁺ after ESI. Collision-induced dissociation of these ions creates product ions at <i>m</i>/<i>z</i> 187 and <i>m</i>/<i>z</i> 169 from cleavage of the glycosidic bond and are present for all disaccharides studied. Both of these product ions were found to adduct water after their formation in a quadrupole ion trap. The kinetics of this water adduction can be measured by isolating either of the product ions and waiting a short time (<1 s) before mass analysis. Additionally, for both product ions, only a fraction of the ions were able to adduct water. This unreactive fraction was measured along with the reaction rate, and the combination of these two values was found to be unique for each disaccharide. Additionally, after CID, a 1000 ms delay can be added, and the ratios of the resulting products ions of <i>m</i>/<i>z</i> 169, 187, and 205 can be used to distinguish linkage position and anomericity with a single tandem mass spectrometry experiment

Miniature Flow-Through Low-Temperature Plasma Ionization Source for Ambient Ionization of Gases and Aerosols

The design and operation of an inexpensive, miniature low-temperature plasma ion source is detailed. The miniature low-temperature plasma ion source is operated in a “flow-through” configuration, wherein the gaseous or aerosolized analyte, caffeine or pyrolyzed ethyl cellulose, in a carrier gas is used as the plasma gas. In this flow-through configuration, the sensitivity for the caffeine standard and the pyrolysis products of ethyl cellulose is maintained or increased and the reproducibility of the ion source is increased. Changes in the relative intensity of ions from the aerosol produced by pyrolysis of ethyl cellulose are observed in the mass spectrum when the low-temperature plasma ion source is used in the flow-through configuration. Experiments suggest this change in relative intensity is likely due to differences in ionization efficiency rather than increased fragmentation of ethyl cellulose pyrolysis products during ionization. Flow-through low-temperature plasma ionization with the miniature ion source is shown to be a promising technique for the ionization of compounds in gases or aerosol particles

Differential Ion Mobility Spectrometry Coupled to Tandem Mass Spectrometry Enables Targeted Leukemia Antigen Detection

Differential ion mobility spectrometry (DIMS) can be used as a filter to remove undesired background ions from reaching the mass spectrometer. The ability to use DIMS as a filter for known analytes makes DIMS coupled to tandem mass spectrometry (DIMS–MS/MS) a promising technique for the detection of cancer antigens that can be predicted by computational algorithms. In experiments using DIMS–MS/MS that were performed without the use of high-performance liquid chromatography (HPLC), a predicted model antigen, GLR (FLSSANEHL), was detected at a concentration of 10 pM (20 amol) in a mixture containing 94 competing model peptide antigens, each at a concentration of 1 μM. Without DIMS filtering, the GLR peptide was undetectable in the mixture even at 100 nM. Again, without using HPLC, DIMS–MS/MS was used to detect 2 of 3 previously characterized antigens produced by the leukemia cell line U937.A2. Because of its sensitivity, a targeted DIMS–MS/MS methodology can likely be used to probe for predicted cancer antigens from cancer cell lines as well as human tumor samples

Distinguishing Biologically Relevant Hexoses by Water Adduction to the Lithium-Cationized Molecule

A method to distinguish the four most common biologically relevant underivatized hexoses, d-glucose, d-galactose, d-mannose, and d-fructose, using only mass spectrometry with no prior separation/derivatization step has been developed. Electrospray of a solution containing hexose and a lithium salt generates [Hexose+Li]⁺. The lithium-cationized hexoses adduct water in a quadrupole ion trap. The rate of this water adduction reaction can be used to distinguish the four hexoses. Additionally, for each hexose, multiple lithiation sites are possible, allowing for multiple structures of [Hexose+Li]⁺. Electrospray produces at least one structure that reacts with water and at least one that does not. The ratio of unreactive lithium-cationized hexose to total lithium-cationized hexose is unique for the four hexoses studied, providing a second method for distinguishing the isomers. Use of the water adduction reaction rate or the unreactive ratio provides two separate methods for confidently (<i>p</i> ≤ 0.02) distinguishing the most common biologically relevant hexoses using only femtomoles of hexose. Additionally, binary mixtures of glucose and fructose were studied. A calibration curve was created by measuring the reaction rate of various samples with different ratios of fructose and glucose. The calibration curve was used to accurately measure the percentage of fructose in three samples of high fructose corn syrup (<4% error)

Distinguishing Biologically Relevant Hexoses by Water Adduction to the Lithium-Cationized Molecule

A method to distinguish the four most common biologically relevant underivatized hexoses, d-glucose, d-galactose, d-mannose, and d-fructose, using only mass spectrometry with no prior separation/derivatization step has been developed. Electrospray of a solution containing hexose and a lithium salt generates [Hexose+Li]⁺. The lithium-cationized hexoses adduct water in a quadrupole ion trap. The rate of this water adduction reaction can be used to distinguish the four hexoses. Additionally, for each hexose, multiple lithiation sites are possible, allowing for multiple structures of [Hexose+Li]⁺. Electrospray produces at least one structure that reacts with water and at least one that does not. The ratio of unreactive lithium-cationized hexose to total lithium-cationized hexose is unique for the four hexoses studied, providing a second method for distinguishing the isomers. Use of the water adduction reaction rate or the unreactive ratio provides two separate methods for confidently (<i>p</i> ≤ 0.02) distinguishing the most common biologically relevant hexoses using only femtomoles of hexose. Additionally, binary mixtures of glucose and fructose were studied. A calibration curve was created by measuring the reaction rate of various samples with different ratios of fructose and glucose. The calibration curve was used to accurately measure the percentage of fructose in three samples of high fructose corn syrup (<4% error)

DiffN Selection of Tandem Mass Spectrometry Precursors

Current data-dependent acquisition (DDA) approaches select precursor ions for tandem mass spectrometry (MS/MS) characterization based on their absolute intensity, known as a TopN approach. Low-abundance species may not be identified as biomarkers in a TopN approach. Herein, a new DDA approach is proposed, DiffN, which uses the relative differential intensity of ions between two samples to selectively target species undergoing the largest fold changes for MS/MS. Using a dual nano-electrospray (nESI) ionization source which allows samples contained in separate capillaries to be analyzed in parallel, the DiffN approach was developed and validated with well-defined lipid extracts. A dual nESI source and DiffN DDA approach was applied to quantify the differences in lipid abundance between two colorectal cancer cell lines. The SW480 and SW620 lines represent a matched pair from the same patient: the SW480 cells from a primary tumor and the SW620 cells from a metastatic lesion. A comparison of TopN and DiffN DDA approaches on these cancer cell samples highlights the ability of DiffN to increase the likelihood of biomarker discovery and the decreased probability of TopN to efficiently select lipid species that undergo large fold changes. The ability of the DiffN approach to efficiently select precursor ions of interest makes it a strong candidate for lipidomic analyses. This DiffN DDA approach may also apply to other molecule classes (e.g., other metabolites or proteins) that are amenable to shotgun analyses

Evaluation of e-liquid toxicity using an open-source high-throughput screening assay

<div><p>The e-liquids used in electronic cigarettes (E-cigs) consist of propylene glycol (PG), vegetable glycerin (VG), nicotine, and chemical additives for flavoring. There are currently over 7,700 e-liquid flavors available, and while some have been tested for toxicity in the laboratory, most have not. Here, we developed a 3-phase, 384-well, plate-based, high-throughput screening (HTS) assay to rapidly triage and validate the toxicity of multiple e-liquids. Our data demonstrated that the PG/VG vehicle adversely affected cell viability and that a large number of e-liquids were more toxic than PG/VG. We also performed gas chromatography–mass spectrometry (GC-MS) analysis on all tested e-liquids. Subsequent nonmetric multidimensional scaling (NMDS) analysis revealed that e-liquids are an extremely heterogeneous group. Furthermore, these data indicated that (i) the more chemicals contained in an e-liquid, the more toxic it was likely to be and (ii) the presence of vanillin was associated with higher toxicity values. Further analysis of common constituents by electron ionization revealed that the concentration of cinnamaldehyde and vanillin, but not triacetin, correlated with toxicity. We have also developed a publicly available searchable website (<a href="http://www.eliquidinfo.org" target="_blank">www.eliquidinfo.org</a>). Given the large numbers of available e-liquids, this website will serve as a resource to facilitate dissemination of this information. Our data suggest that an HTS approach to evaluate the toxicity of multiple e-liquids is feasible. Such an approach may serve as a roadmap to enable bodies such as the Food and Drug Administration (FDA) to better regulate e-liquid composition.</p></div

Vanillin and cinnamaldehyde concentrations correlate with toxicity in select e-liquids.

<p>(A) Graph showing toxicity (LC₅₀) versus “vanillin” in select e-liquids. LC₅₀ = −2.70 × log₁₀(vanillin [M]) + 5.06; R² = 0.62 (linear regression analysis). (B) Graph showing toxicity (LC₅₀) versus “cinnamaldehyde” in select e-liquids. LC₅₀ = −1.12 × log₁₀(cinnamaldehyde[M]) + 1.08; R² = 0.75 (linear regression analysis). (C) Graph showing toxicity (LC₅₀) versus “triacetin” in select e-liquids. “N.B.” = no linear relationship was detected for triacetin. We used this chemical as an example of nontoxic control. Raw data are available in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2003904#pbio.2003904.s010" target="_blank">S7 Data</a>. LC₅₀, concentration at which a given agent is lethal to 50% of the cells; nc, no nicotine.</p

Toxicity of “vaped” versus neat e-liquids.

<p>(A) Mean normalized viability of HEK293T cells following exposure of vaped e-liquids. <i>N</i> ≥ 5 per treatment. (B) Mean normalized viability of primary human alveolar macrophages following exposure of vaped e-liquids. <i>N</i> ≥ 5 per treatment. (C) Mean normalized viability of HBECs following exposure to vaped e-liquids. <i>N</i> ≥ 5 per treatment. (D) Graph showing HEK293T vaped viability versus HEK293T toxicity (LC₅₀) obtained using neat e-liquids. Linear regression R² = 0.66. (E) Graph showing primary human alveolar macrophage vaped viability versus HEK293T toxicity (LC₅₀). Linear regression R² = 0.06. (F) HBEC viability using vaped e-liquids versus HEK293T toxicity (LC₅₀). Linear regression R² = 0.74. * = <i>p</i> < 0.05 different from control. For A, B, and C we performed statistical analysis using one-way ANOVA followed by Dunnett’s Test. B. N. B. Smoothie, Chocolate B., and Coconut Water. Raw data are available in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2003904#pbio.2003904.s008" target="_blank">S5 Data</a>. B. N. B. Smoothie, Banana Nut Bread Smoothie; Chocolate B., Chocolate Banana; HBEC, human bronchial epithelial cells; HEK293T, human embryonic kidney 293 cells; LC₅₀, concentration at which a given agent is lethal to 50% of the cells.</p