Publications by authors named "Gary L Glish"

54 Publications

Cigarillos Compromise the Mucosal Barrier and Protein Expression in Airway Epithelia.

Am J Respir Cell Mol Biol 2020 12;63(6):767-779

Department of Pathology and Laboratory Medicine.

Smoking remains a leading cause of preventable morbidity and mortality worldwide. Despite a downward trend in cigarette use, less-regulated tobacco products, such as cigarillos, which are often flavored to appeal to specific demographics, such as younger people, are becoming increasingly popular. Cigar/cigarillo smoking has been considered a safer alternative to cigarettes; however, the health risks associated with cigar in comparison with cigarette smoking are not well understood. To address this knowledge gap, we characterized the effects of multiple brands of cigarillos on the airway epithelium using and models. To analyze these effects, we assessed the cellular viability and integrity of smoke-exposed primary airway cell cultures. We also investigated the protein compositions of apical secretions from cigarillo-exposed airway epithelial cultures and BAL fluid of cigarillo-exposed mice through label-free quantitative proteomics and determined the chemical composition of smoke collected from the investigated cigarillo products. We found that cigarillo smoke exerts similar or greater effects than cigarette smoke in terms of reduced cell viability; altered protein levels, including those of innate immune proteins; induced oxidative-stress markers; and greater nicotine delivery to cells. The analysis of the chemical composition of the investigated cigarillo products revealed differences that might be linked to the differential effects of these products on cell viability and protein abundance profiles, which have been associated with a range of health risks in the context of airway biology. These findings contradict the assumption that cigarillos might be safer and less harmful than cigarettes. Instead, our results indicate that cigarillo smoke is associated with equal or greater health risks and the same or increased airway toxicity compared with cigarette smoke.
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http://dx.doi.org/10.1165/rcmb.2019-0085OCDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7790145PMC
December 2020

Alkali Metal Cationization of Tumor-associated Antigen Peptides for Improved Dissociation and Measurement by Differential Ion Mobility-Mass Spectrometry.

J Proteome Res 2020 08 20;19(8):3176-3183. Epub 2020 Jul 20.

Department of Chemistry, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, United States.

Tandem mass spectrometry (MS/MS) is a highly sensitive and selective method for the detection of tumor-associated peptide antigens. These short, nontryptic sequences may lack basic residues, resulting in the formation of predominantly [peptide + H] ions in electrospray. These singly charged ions tend to undergo inefficient dissociation, leading to issues in sequence determination. Addition of alkali metal salts to the electrospray solvent can drive the formation of [peptide + H + metal] ions that have enhanced dissociation characteristics relative to [peptide + H] ions. Both previously identified tumor-associated antigens and predicted neoantigen sequences were investigated. The previously reported rearrangement mechanism in MS/MS of sodium-cationized peptides is applied here to demonstrate complete C-terminal sequencing of tumor-associated peptide antigens. Differential ion mobility spectrometry (DIMS) is shown to selectively enrich [peptide + H + metal] species by filtering out singly charged interferences at relatively low field strengths, offsetting the decrease in signal intensity associated with the use of alkali metal cations.
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http://dx.doi.org/10.1021/acs.jproteome.0c00157DOI Listing
August 2020

Flavored e-liquids increase cytoplasmic Ca levels in airway epithelia.

Am J Physiol Lung Cell Mol Physiol 2020 02 6;318(2):L226-L241. Epub 2019 Nov 6.

Marsico Lung Institute, University of North Carolina, Chapel Hill, North Carolina.

E-cigarettes are noncombustible, electronic nicotine-delivery devices that aerosolize an e-liquid, i.e., nicotine, in a propylene glycol-vegetable glycerin vehicle that also contains flavors. While the effects of nicotine are relatively well understood, more information regarding the potential biological effects of the other e-liquid constituents is needed. This is a serious concern, because e-liquids are available in >7,000 distinct flavors. We previously demonstrated that many e-liquids affect cell growth/viability through an unknown mechanism. Since Ca is a ubiquitous second messenger that regulates cell growth, we characterized the effects of e-liquids on cellular Ca homeostasis. To better understand the extent of this effect, we screened e-liquids for their ability to alter cytosolic Ca levels and found that 42 of 100 flavored e-liquids elicited a cellular Ca response. Banana Pudding (BP) e-liquid, a representative e-liquid from this group, caused phospholipase C activation, endoplasmic reticulum (ER) Ca release, store-operated Ca entry (SOCE), and protein kinase C (PKCα) phosphorylation. However, longer exposures to BP e-liquid depleted ER Ca stores and inhibited SOCE, suggesting that this e-liquid may alter Ca homeostasis by short- and long-term mechanisms. Since dysregulation of Ca signaling can cause chronic inflammation, ER stress, and abnormal cell growth, flavored e-cigarette products that can elicit cell Ca responses should be further screened for potential toxicity.
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http://dx.doi.org/10.1152/ajplung.00123.2019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7052665PMC
February 2020

Computational modeling and confirmation of leukemia-associated minor histocompatibility antigens.

Blood Adv 2018 08;2(16):2052-2062

Lineberger Comprehensive Cancer Center.

T-cell responses to minor histocompatibility antigens (mHAs) mediate both antitumor immunity (graft-versus-leukemia [GVL]) and graft-versus-host disease (GVHD) in allogeneic stem cell transplant. Identifying mHAs with high allele frequency, tight binding affinity to common HLA molecules, and narrow tissue restriction could enhance immunotherapy against leukemia. Genotyping and HLA allele data from 101 HLA-matched donor-recipient pairs (DRPs) were computationally analyzed to predict both class I and class II mHAs likely to induce either GVL or GVHD. Roughly twice as many mHAs were predicted in HLA-matched unrelated donor (MUD) stem cell transplantation (SCT) compared with HLA-matched related transplants, an expected result given greater genetic disparity in MUD SCT. Computational analysis predicted 14 of 18 previously identified mHAs, with 2 minor antigen mismatches not being contained in the patient cohort, 1 missed mHA resulting from a noncanonical translation of the peptide antigen, and 1 case of poor binding prediction. A predicted peptide epitope derived from GRK4, a protein expressed in acute myeloid leukemia and testis, was confirmed by targeted differential ion mobility spectrometry-tandem mass spectrometry. T cells specific to UNC-GRK4-V were identified by tetramer analysis both in DRPs where a minor antigen mismatch was predicted and in DRPs where the donor contained the allele encoding UNC-GRK4-V, suggesting that this antigen could be both an mHA and a cancer-testis antigen. Computational analysis of genomic and transcriptomic data can reliably predict leukemia-associated mHA and can be used to guide targeted mHA discovery.
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http://dx.doi.org/10.1182/bloodadvances.2018022475DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6113610PMC
August 2018

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

Anal Chem 2018 08 23;90(15):9117-9124. Epub 2018 Jul 23.

Department of Chemistry , The University of North Carolina at Chapel Hill , Chapel Hill , North Carolina 27599 United States.

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.
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http://dx.doi.org/10.1021/acs.analchem.8b01528DOI Listing
August 2018

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

PLoS Biol 2018 03 27;16(3):e2003904. Epub 2018 Mar 27.

Marsico Lung Institute/Cystic Fibrosis Research Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina.

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 (www.eliquidinfo.org). 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.
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http://dx.doi.org/10.1371/journal.pbio.2003904DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5870948PMC
March 2018

Paper Spray Mass Spectrometry for High-Throughput Quantification of Nicotine and Cotinine.

Anal Methods 2018 28;10(1):46-50. Epub 2017 Nov 28.

Department of Chemistry, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599 USA.

The rapid release of new tobacco products requires high-throughput quantitative methods to support tobacco research. Sample preparation for LC-MS and GC-MS is time consuming and limits throughput. Paper spray tandem mass spectrometry (PS-MS/MS) is proposed and validated as a simple and rapid method for quantification of nicotine and cotinine in complex matrices to support tobacco-related research. Air liquid interface (ALI) human tracheobronchial epithelial cell (HTBEC) cultures were exposed to tobacco smoke using a Vitrocell VC-10 smoking machine. Apical culture washes (phosphate buffered saline, PBS) and basolateral media were analyzed with the PS-MS/MS method. GC-MS/MS was used as a comparative quantitative technique. The PS-MS/MS approach allowed for direct spotting of samples on the paper substrate, whereas the GC-MS/MS method required additional sample preparation in the form of solvent-solvent extraction. Limits of quantitation (LOQs) were higher with the PS-MS/MS approach than GC-MS/MS, but still below the relevant concentrations found in HTBEC smoke exposure experiments as well as most clinical applications. PS-MS/MS is readily achieved on mass spectrometers that include atmospheric pressure inlets, and allows for convenient quantification from complex matrices that would otherwise require additional sample preparation and chromatographic separation.
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http://dx.doi.org/10.1039/C7AY02204BDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5858713PMC
November 2017

Resolution and Assignment of Differential Ion Mobility Spectra of Sarcosine and Isomers.

J Am Soc Mass Spectrom 2018 04 21;29(4):752-760. Epub 2018 Feb 21.

Laboratoire de Chimie Physique, Bâtiment 349, Université Paris-Sud, CNRS, Université Paris-Saclay, F-91405, Orsay, France.

Due to their central role in biochemical processes, fast separation and identification of amino acids (AA) is of importance in many areas of the biomedical field including the diagnosis and monitoring of inborn errors of metabolism and biomarker discovery. Due to the large number of AA together with their isomers and isobars, common methods of AA analysis are tedious and time-consuming because they include a chromatographic separation step requiring pre- or post-column derivatization. Here, we propose a rapid method of separation and identification of sarcosine, a biomarker candidate of prostate cancer, from isomers using differential ion mobility spectrometry (DIMS) interfaced with a tandem mass spectrometer (MS/MS) instrument. Baseline separation of protonated sarcosine from α- and β-alanine isomers can be easily achieved. Identification of DIMS peak is performed using an isomer-specific activation mode where DIMS- and mass-selected ions are irradiated at selected wavenumbers allowing for the specific fragmentation via an infrared multiple photon dissociation (IRMPD) process. Two orthogonal methods to MS/MS are thus added, where the MS/MS(IRMPD) is nothing but an isomer-specific multiple reaction monitoring (MRM) method. The identification relies on the comparison of DIMS-MS/MS(IRMPD) chromatograms recorded at different wavenumbers. Based on the comparison of IR spectra of the three isomers, it is shown that specific depletion of the two protonated α- and β-alanine can be achieved, thus allowing for clear identification of the sarcosine peak. It is also demonstrated that DIMS-MS/MS(IRMPD) spectra in the carboxylic C=O stretching region allow for the resolution of overlapping DIMS peaks. Graphical Abstract ᅟ.
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http://dx.doi.org/10.1007/s13361-018-1902-5DOI Listing
April 2018

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

Anal Chem 2018 02 16;90(3):2048-2054. Epub 2018 Jan 16.

Department of Chemistry, Caudill Laboratories, The University of North Carolina at Chapel Hill , Chapel Hill, North Carolina 27599-3290, United States.

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 m/z 187 and m/z 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 m/z 169, 187, and 205 can be used to distinguish linkage position and anomericity with a single tandem mass spectrometry experiment.
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http://dx.doi.org/10.1021/acs.analchem.7b04162DOI Listing
February 2018

Use of an Open Port Sampling Interface Coupled to Electrospray Ionization for the On-Line Analysis of Organic Aerosol Particles.

J Am Soc Mass Spectrom 2018 02 11;29(2):297-303. Epub 2017 Sep 11.

Department of Chemistry, Caudill Laboratories, The University of North Carolina at Chapel Hill, Chapel Hill, NC, 27599-3290, USA.

A simple design for an open port sampling interface coupled to electrospray ionization (OPSI-ESI) is presented for the analysis of organic aerosols. The design uses minimal modifications to a Bruker electrospray (ESI) emitter to create a continuous flow, self-aspirating open port sampling interface. Considerations are presented for introducing aerosol to the open port sampling interface including aerosol gas flow and solvent flow rates. The device has been demonstrated for use with an aerosol of nicotine as well as aerosol formed in the pyrolysis of biomass. Upon comparison with extractive electrospray ionization (EESI), this device has similar sensitivity with increased reproducibility by nearly a factor of three. The device has the form factor of a standard Bruker/Agilent ESI emitter and can be used without any further instrument modifications. Graphical Abstract ᅟ.
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http://dx.doi.org/10.1007/s13361-017-1776-yDOI Listing
February 2018

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

Anal Chem 2017 10 20;89(19):10504-10510. Epub 2017 Sep 20.

Department of Chemistry, Caudill Laboratories, The University of North Carolina at Chapel Hill , Chapel Hill, North Carolina 27599-3290, United States.

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 (p ≤ 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).
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http://dx.doi.org/10.1021/acs.analchem.7b02647DOI Listing
October 2017

Variables Affecting the Internal Energy of Peptide Ions During Separation by Differential Ion Mobility Spectrometry.

J Am Soc Mass Spectrom 2017 10 26;28(10):2160-2169. Epub 2017 Jun 26.

Department of Chemistry, Caudill Laboratories, The University of North Carolina at Chapel Hill, Campus Box 3290, Chapel Hill, NC, 27599-3290, USA.

Differential ion mobility spectrometry (DIMS) devices separate ions on the basis of differences in ion mobility in low and high electric fields, and can be used as a stand-alone analytical method or as a separation step before further analysis. As with other ion mobility separation techniques, the ability of DIMS separations to retain the structural characteristics of analytes has been of concern. For DIMS separations, this potential loss of ion structure originates from the fact that the separations occur at atmospheric pressure and the ions, during their transit through the device, undergo repeated collisions with the DIMS carrier gas while being accelerated by the electric field. These collisions have the ability to increase the internal energy distribution of the ions, which can cause isomerization or fragmentation. The increase in internal energy of the ions is based on a number of variables, including the dispersion field and characteristics of the carrier gas such as temperature and composition. The effects of these parameters on the intra-DIMS fragmentation of multiply charged ions of the peptides bradykinin (RPPGFSPFR) and GLISH are discussed herein. Furthermore, similarities and differences in the internal energy deposition that occur during collisional activation in tandem mass spectrometry experiments are discussed, as the fragmentation pathways accessed by both are similar. Graphical Abstract ᅟ.
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http://dx.doi.org/10.1007/s13361-017-1726-8DOI Listing
October 2017

Flavored e-cigarette liquids and cinnamaldehyde impair respiratory innate immune cell function.

Am J Physiol Lung Cell Mol Physiol 2017 08 11;313(2):L278-L292. Epub 2017 May 11.

Curriculum in Toxicology, School of Medicine, University of North Carolina, Chapel Hill, North Carolina;

Innate immune cells of the respiratory tract are the first line of defense against pathogenic and environmental insults. Failure of these cells to perform their immune functions leaves the host susceptible to infection and may contribute to impaired resolution of inflammation. While combustible tobacco cigarettes have been shown to suppress respiratory immune cell function, the effects of flavored electronic cigarette liquids (e-liquids) and individual flavoring agents on respiratory immune cell responses are unknown. We investigated the effects of seven flavored nicotine-free e-liquids on primary human alveolar macrophages, neutrophils, and natural killer (NK) cells. Cells were challenged with a range of e-liquid dilutions and assayed for their functional responses to pathogenic stimuli. End points included phagocytic capacity (neutrophils and macrophages), neutrophil extracellular trap formation, proinflammatory cytokine production, and cell-mediated cytotoxic response (NK cells). E-liquids were then analyzed via mass spectrometry to identify individual flavoring components. Three cinnamaldehyde-containing e-liquids exhibited dose-dependent broadly immunosuppressive effects. Quantitative mass spectrometry was used to determine concentrations of cinnamaldehyde in each of the three e-liquids, and cells were subsequently challenged with a range of cinnamaldehyde concentrations. Cinnamaldehyde alone recapitulated the impaired function observed with e-liquid exposures, and cinnamaldehyde-induced suppression of macrophage phagocytosis was reversed by addition of the small-molecule reducing agent 1,4-dithiothreitol. We conclude that cinnamaldehyde has the potential to impair respiratory immune cell function, illustrating an immediate need for further toxicological evaluation of chemical flavoring agents to inform regulation governing their use in e-liquid formulations.
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http://dx.doi.org/10.1152/ajplung.00452.2016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5582929PMC
August 2017

Little Cigars are More Toxic than Cigarettes and Uniquely Change the Airway Gene and Protein Expression.

Sci Rep 2017 04 27;7:46239. Epub 2017 Apr 27.

Marsico Lung Institute, The University of North Carolina at Chapel Hill, NC, 27599, USA.

Little cigars (LCs) are regulated differently than cigarettes, allowing them to be potentially targeted at youth/young adults. We exposed human bronchial epithelial cultures (HBECs) to air or whole tobacco smoke from cigarettes vs. LCs. Chronic smoke exposure increased the number of dead cells, lactate dehydrogenase release, and interleukin-8 (IL-8) secretion and decreased apical cilia, cystic fibrosis transmembrane conductance regulator (CFTR) protein levels, and transepithelial resistance. These adverse effects were significantly greater in LC-exposed HBECs than cigarette exposed cultures. LC-exposure also elicited unique gene expression changes and altered the proteomic profiles of airway apical secretions compared to cigarette-exposed HBECs. Gas chromatography-mass spectrometry (GC-MS) analysis indicated that LCs produced more chemicals than cigarettes, suggesting that the increased chemical load of LCs may be the cause of the greater toxicity. This is the first study of the biological effects of LCs on pulmonary epithelia and our observations strongly suggest that LCs pose a more severe danger to human health than cigarettes.
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http://dx.doi.org/10.1038/srep46239DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5406835PMC
April 2017

Flavored e-cigarette liquids reduce proliferation and viability in the CALU3 airway epithelial cell line.

Am J Physiol Lung Cell Mol Physiol 2017 07 20;313(1):L52-L66. Epub 2017 Apr 20.

Marsico Lung Institute, The University of North Carolina, Chapel Hill, North Carolina;

E-cigarettes are generally thought of as a safer smoking alternative to traditional cigarettes. However, little is known about the effects of e-cigarette liquids (e-liquids) on the lung. Since over 7,000 unique flavors have been identified for purchase in the United States, our goal was to conduct a screen that would test whether different flavored e-liquids exhibited different toxicant profiles. We tested the effects of 13 different flavored e-liquids [with nicotine and propylene glycol/vegetable glycerin (PG/VG) serving as controls] on a lung epithelial cell line (CALU3). Using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay as an indicator of cell proliferation/viability, we demonstrated a dose-dependent decrease of MTT metabolism by all flavors tested. However, a group of four flavors consistently showed significantly greater toxicity compared with the PG/VG control, indicating the potential for some flavors to elicit more harmful effects than others. We also tested the aerosolized "vapor" from select e-liquids on cells and found similar dose-dependent trends, suggesting that direct e-liquid exposures are a justifiable first-pass screening approach for determining relative e-liquid toxicity. We then identified individual chemical constituents for all 13 flavors using gas chromatography-mass spectrometry. These data revealed that beyond nicotine and PG/VG, the 13 flavored e-liquids have diverse chemical constituents. Since all of the flavors exhibited some degree of toxicity and a diverse array of chemical constituents with little inhalation toxicity available, we conclude that flavored e-liquids should be extensively tested on a case-by-case basis to determine the potential for toxicity in the lung and elsewhere.
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http://dx.doi.org/10.1152/ajplung.00392.2016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5538872PMC
July 2017

Identifying the D-Pentoses Using Water Adduction to Lithium Cationized Molecule.

J Am Soc Mass Spectrom 2017 07 14;28(7):1420-1424. Epub 2017 Apr 14.

Department of Chemistry, Caudill Laboratories, The University of North Carolina at Chapel Hill, Chapel Hill, NC, 27599-3290, USA.

A method has been developed that is capable of distinguishing an exhaustive list of underivatized D-pentoses with only a mass spectrometer. Electrospray ionization (ESI) of a solution containing a pentose and a lithium salt yields [Pentose + Li]. These lithiated pentoses adduct water in a quadrupole ion trap. The reaction rate of water adduction is unique for several of the pentose isomers. Additionally, there are multiple potential gas-phase lithiation sites to form [Pentose + Li]. A mixture of ions with at least one reactive (water adducting) and at least one unreactive (non-adducting) lithiation site is formed for each pentose. The water adduction reaction rate along with the unreactive fraction of lithiated pentose can be used to completely discriminate all D-pentoses. Graphical Abstract ᅟ.
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http://dx.doi.org/10.1007/s13361-017-1656-5DOI Listing
July 2017

Metal Cationization Extractive Electrospray Ionization Mass Spectrometry of Compounds Containing Multiple Oxygens.

J Am Soc Mass Spectrom 2017 06 28;28(6):1030-1035. Epub 2016 Nov 28.

Department of Chemistry, Caudill Laboratories, The University of North Carolina at Chapel Hill, Chapel Hill, NC, 27599-3290, USA.

Extractive electrospray ionization is an ambient ionization technique that allows real-time sampling of liquid samples, including organic aerosols. Similar to electrospray ionization, the composition of the electrospray solvent used in extractive electrospray ionization can easily be altered to form metal cationized molecules during ionization simply by adding a metal salt to the electrospray solvent. An increase in sensitivity is observed for some molecules that are lithium, sodium, or silver cationized compared with the protonated molecule formed in extractive electrospray ionization with an acid additive. Tandem mass spectrometry of metal cationized molecules can also significantly improve the ability to identify a compound. Tandem mass spectrometry of lithium and silver cationized molecules can result in an increase in the number and uniqueness of dissociation pathways relative to [M + H]. These results highlight the potential for extractive electrospray ionization with metal cationization in analyzing complex aerosol mixtures. Graphical Abstract ᅟ.
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http://dx.doi.org/10.1007/s13361-016-1546-2DOI Listing
June 2017

Increased Ion Transmission for Differential Ion Mobility Combined with Mass Spectrometry by Implementation of a Flared Inlet Capillary.

J Am Soc Mass Spectrom 2017 Jan 17;28(1):119-124. Epub 2016 Oct 17.

Department of Chemistry, Caudill Laboratories, The University of North Carolina at Chapel Hill, Chapel Hill, NC, 27599-3290, USA.

Differential ion mobility spectrometry (DIMS) is capable of separating components of complex mixtures prior to mass spectrometric analysis, thereby increasing signal-to-noise and signal-to-background ratios on millisecond timescales. However, adding a DIMS device to the front end of a mass spectrometer can reduce the signal intensity of subsequent mass spectrometric analysis. This is a result, in part, of ions lost due to inefficient transfer of ions from the DIMS device through the aperture leading into the mass spectrometer. This problem of transferring ions can be at least partially corrected by modifying the front end of the inlet capillary leading to the vacuum of the mass spectrometer. The inner diameter of the ion-sampling end of the inlet capillary was enlarged by drilling into the face. This results in a conical flare at the front end of the capillary, while the other end of the capillary remains unmodified. These flared capillaries allow for a greater number of ions from the DIMS device to be sampled relative to the unmodified standard capillary. Four flare dimensions were tested, differing by the angle between the wall of the flare and the outer wall of the inlet capillary. All flared capillaries showed greater signal intensity than the standard capillary with a DIMS device present without reducing the resolving power. It was also observed that the signal intensity increased as the flare angle decreased. The flared capillary with the smallest flare angle showed greater than a fivefold increase in signal intensity compared with the standard capillary. Graphical Abstract ᅟ.
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http://dx.doi.org/10.1007/s13361-016-1509-7DOI Listing
January 2017

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

Anal Chem 2015 Dec 18;87(23):11887-92. Epub 2015 Nov 18.

University of North Carolina at Chapel Hill , Department of Chemistry, Chapel Hill, North Carolina 27599-3290, United States.

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.
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http://dx.doi.org/10.1021/acs.analchem.5b03447DOI Listing
December 2015

Resolving powers of >7900 using linked scans: how well does resolving power describe the separation capability of differential ion mobility spectrometry.

Analyst 2015 Oct;140(20):6871-8

Department of Chemistry, Caudill and Kenan Laboratories, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-3290, USA.

Differential ion mobility spectrometry (DIMS) separations are described using similar terminology to liquid chromatography, capillary electrophoresis, and drift tube ion mobility spectrometry. The characterization and comparison of all these separations are typically explained in terms of resolving power, resolution, and/or peak capacity. A major difference between these separations is that DIMS separations are in space whereas the others are separations in time. However, whereas separations in time can, in theory, be extended infinitely, separations in space, such as DIMS separations, are constrained by the physical dimensions of the device. One method to increase resolving power of DIMS separations is to use helium in the DIMS carrier gas. However, ions have a greater mobility in helium which causes more ions to be neutralized due to collisions with the DIMS electrodes or electrode housing, i.e. the space constraints. This neutralization of ions can lead to the loss of an entire peak, or peaks, from a DIMS scan. To take advantage of the benefits of helium use while reducing ion losses, linked scans were developed. During a linked scan the amount of helium present in the DIMS carrier gas is decreased as the compensation field is increased. A comparison of linked scans to compensation field scans with constant helium is presented herein. Resolving powers >7900 are obtained with linked scans. However, this result highlights the limitation of using resolving power as a metric to describe DIMS separations.
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http://dx.doi.org/10.1039/c5an00845jDOI Listing
October 2015

Improved Differential Ion Mobility Separations Using Linked Scans of Carrier Gas Composition and Compensation Field.

J Am Soc Mass Spectrom 2015 Oct 7;26(10):1746-53. Epub 2015 Jul 7.

Department of Chemistry, Caudill and Kenan Laboratories, The University of North Carolina at Chapel Hill, Chapel Hill, NC, 27599-3290, USA.

Differential ion mobility spectrometry (DIMS) separates ions based on differences in their mobilities in low and high electric fields. When coupled to mass spectrometric analyses, DIMS has the ability to improve signal-to-background by eliminating isobaric and isomeric compounds for analytes in complex mixtures. DIMS separation power, often measured by resolution and peak capacity, can be improved through increasing the fraction of helium in the nitrogen carrier gas. However, because the mobility of ions is higher in helium, a greater number of ions collide with the DIMS electrodes or housing, yielding losses in signal intensity. To take advantage of the benefits of helium addition on DIMS separations and reduce ion losses, linked scans were developed. In a linked scan the helium content of the carrier gas is reduced as the compensation field is increased. Linked scans were compared with conventional compensation field scans with constant helium content for the protein ubiquitin and a tryptic digest of bovine serum albumin (BSA). Linked scans yield better separation of ubiquitin charge states and enhanced peak capacities for the analysis of BSA compared with compensation field scans with constant helium carrier gas percentages. Linked scans also offer improved signal intensity retention in comparison to compensation field scans with constant helium percentages in the carrier gas.
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http://dx.doi.org/10.1007/s13361-015-1208-9DOI Listing
October 2015

Probing Mobility-Selected Saccharide Isomers: Selective Ion-Molecule Reactions and Wavelength-Specific IR Activation.

J Phys Chem A 2015 Jun 15;119(23):6057-64. Epub 2015 Apr 15.

†Laboratoire de Chimie Physique, Université Paris Sud, 91400 Orsay, France.

Differential Ion Mobility Spectrometry (DIMS) provides orthogonal separation to mass spectrometry, and DIMS combined with the high sensitivity of a quadrupole ion-trap is shown to be useful for the separation and identification of saccharides. A comprehensive analysis of the separation of anomers (α- and β-methylated glucose) and epimers (α-methylated glucose and mannose) ionized with Li(+), Na(+), and K(+) is performed. DIMS separation is found to be better for saccharides cationized with the two latter species. The corresponding resolving power for the two glucose anomers with Na(+) is found to be very close to the corresponding drift-tube IMS value. The lithiated complexes are investigated further using a combination of infrared spectroscopy integrated to ion-trap mass spectrometry and quantum chemical calculations. Together with DIMS, consistent results are obtained. It is found that two competing structural motifs might be at play, depending on the subtle balance between the maximization of the coordination of the metal cation and the intrinsic conformational energetics of the saccharide, which is for a large part driven by hydrogen bonding. The comparison of simulated and observed spectra clearly shows that a band at ∼3400 cm(-1) is specific to a structural motif found in the lithiated glucose complexes, which could explain the trends observed in the DIMS spectra of the saccharide complexes. It is shown that DIMS-MS/MS using wavelength specific IR activation would provide a new orthogonal dimension to mass spectrometry.
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http://dx.doi.org/10.1021/jp511975fDOI Listing
June 2015

Low-temperature plasma ionization-mass spectrometry for the analysis of compounds in organic aerosol particles.

Anal Chem 2015 Feb 28;87(4):2249-54. Epub 2015 Jan 28.

Department of Chemistry, University of North Carolina at Chapel Hill , Chapel Hill, North Carolina 27599-3290, United States.

Low-temperature plasma ionization, a technique that causes minimal fragmentation during ionization, is investigated as an ionization technique for mass spectrometric detection of the compounds in ambient organic aerosols in real time. The experiments presented in this paper demonstrate that ions are generated from compounds in the aerosol particles. The utility of this technique for detection of both positive and negative ions from the pyrolysate of multiple natural polymers is presented. Ultimately, low-temperature plasma ionization is shown to be a promising ionization technique for detection of compounds in organic aerosols by mass spectrometry.
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http://dx.doi.org/10.1021/ac5038889DOI Listing
February 2015

Peptide/MHC tetramer-based sorting of CD8⁺ T cells to a leukemia antigen yields clonotypes drawn nonspecifically from an underlying restricted repertoire.

Cancer Immunol Res 2015 Mar 9;3(3):228-35. Epub 2015 Jan 9.

Lineberger Comprehensive Cancer Center, Chapel Hill, North Carolina. Department of Medicine, University of North Carolina, Chapel Hill, North Carolina.

Testing of T cell-based cancer therapeutics often involves measuring cancer antigen-specific T-cell populations with the assumption that they arise from in vivo clonal expansion. This analysis, using peptide/MHC tetramers, is often ambiguous. From a leukemia cell line, we identified a CDK4-derived peptide epitope, UNC-CDK4-1 (ALTPVVVTL), that bound HLA-A*02:01 with high affinity and could induce CD8⁺ T-cell responses in vitro. We identified UNC-CDK4-1/HLA-A*02:01 tetramer⁺ populations in 3 of 6 patients with acute myeloid leukemia who had undergone allogeneic stem cell transplantation. Using tetramer-based, single-cell sorting and T-cell receptor β (TCRβ) sequencing, we identified recurrent UNC-CDK4-1 tetramer-associated TCRβ clonotypes in a patient with a UNC-CDK4-1 tetramer⁺ population, suggesting in vivo T-cell expansion to UNC-CDK4-1. In parallel, we measured the patient's TCRβ repertoire and found it to be highly restricted/oligoclonal. The UNC-CDK4-1 tetramer-associated TCRβ clonotypes represented >17% of the entire TCRβ repertoire-far in excess of the UNC-CDK4-1 tetramer⁺ frequency-indicating that the recurrent TCRβ clonotypes identified from UNC-CDK-4-1 tetramer⁺ cells were likely a consequence of the extremely constrained T-cell repertoire in the patient and not in vivo UNC-CDK4-1-driven clonal T-cell expansion. Mapping recurrent TCRβ clonotype sequences onto TCRβ repertoires can help confirm or refute antigen-specific T-cell expansion in vivo.
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http://dx.doi.org/10.1158/2326-6066.CIR-14-0001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4351150PMC
March 2015

Differential ion mobility spectrometry coupled to tandem mass spectrometry enables targeted leukemia antigen detection.

J Proteome Res 2014 Oct 11;13(10):4356-62. Epub 2014 Sep 11.

Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill , 450 West Drive, 21-244, Chapel Hill, North Carolina 27599, United States.

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.
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http://dx.doi.org/10.1021/pr500527cDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4184456PMC
October 2014

Optimization of peptide separations by differential ion mobility spectrometry.

J Am Soc Mass Spectrom 2014 Sep 3;25(9):1592-9. Epub 2014 Jul 3.

Department of Chemistry, Caudill and Kenan Laboratories, The University of North Carolina at Chapel Hill, Chapel Hill, NC, 27599-3290, USA.

Differential ion mobility spectrometry (DIMS) has the ability to separate gas phase ions based on their difference in ion mobility in low and high electric fields. DIMS can be used to separate mixtures of isobaric and isomeric species indistinguishable by mass spectrometry (MS). DIMS can also be used as a filter to improve the signal-to-background of analytes in complex samples. The resolving power of DIMS separations can be improved several ways, including increasing the dispersion field and increasing the amount of helium in the nitrogen carrier gas. It has been previously demonstrated that the addition of helium to the DIMS carrier gas provides improves separations when the dispersion field is the kept constant as helium content is varied. However, helium has a lower breakdown voltage than nitrogen. Therefore, as the percent helium content in the nitrogen carrier gas is increased, the highest dispersion field accessible decreases. This work presents the trade-offs between increasing dispersion fields and using helium in the carrier gas by comparing the separation of a mixture of isobaric peptides. The maximum resolution for a separation of a mixture of three peptides with the same nominal molar mass was achieved by using a high dispersion field (~72 kV/cm) with pure nitrogen as the carrier gas within the DIMS assembly. The conditions used to achieve the maximum resolution also exhibit the lowest ion transmission through the assembly, suggesting that it is necessary to consider the trade-off between sensitivity and resolution when optimizing DIMS conditions for a given application.
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http://dx.doi.org/10.1007/s13361-014-0941-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4458851PMC
September 2014

Cation recombination energy/coulomb repulsion effects in ETD/ECD as revealed by variation of charge per residue at fixed total charge.

J Am Soc Mass Spectrom 2013 Nov 9;24(11):1676-89. Epub 2013 Apr 9.

Department of Chemistry, Purdue University, West Lafayette, IN, 47907-2084, USA.

Electron capture dissociation (ECD) and electron transfer dissociation (ETD) experiments in electrodynamic ion traps operated in the presence of a bath gas in the 1-10 mTorr range have been conducted on a common set of doubly protonated model peptides of the form X(AG)nX (X = lysine, arginine, or histidine, n = 1, 2, or 4). The partitioning of reaction products was measured using thermal electrons, anions of azobenzene, and anions of 1,3-dinitrobenzene as reagents. Variation of n alters the charge per residue of the peptide cation, which affects recombination energy. The ECD experiments showed that H-atom loss is greatest for the n = 1 peptides and decreases as n increases. Proton transfer in ETD, on the other hand, is expected to increase as charge per residue decreases (i.e., as n increases). These opposing tendencies were apparent in the data for the K(AG)nK peptides. H-atom loss appeared to be more prevalent in ECD than in ETD and is rationalized on the basis of either internal energy differences, differences in angular momentum transfer associated with the electron capture versus electron transfer processes, or a combination of the two. The histidine peptides showed the greatest extent of charge reduction without dissociation, the arginine peptides showed the greatest extent of side-chain cleavages, and the lysine peptides generally showed the greatest extent of partitioning into the c/z•-product ion channels. The fragmentation patterns for the complementary c- and z•-ions for ETD and ECD were found to be remarkably similar, particularly for the peptides with X = lysine.
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http://dx.doi.org/10.1007/s13361-013-0606-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3795911PMC
November 2013

A novel HLA-A*0201 restricted peptide derived from cathepsin G is an effective immunotherapeutic target in acute myeloid leukemia.

Clin Cancer Res 2013 Jan 12;19(1):247-57. Epub 2012 Nov 12.

Stem Cell Transplantation and Cellular Therapy, Surgical Oncology, and Melanoma Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA.

Purpose: Immunotherapy targeting aberrantly expressed leukemia-associated antigens has shown promise in the management of acute myeloid leukemia (AML). However, because of the heterogeneity and clonal evolution that is a feature of myeloid leukemia, targeting single peptide epitopes has had limited success, highlighting the need for novel antigen discovery. In this study, we characterize the role of the myeloid azurophil granule protease cathepsin G (CG) as a novel target for AML immunotherapy.

Experimental Design: We used Immune Epitope Database and in vitro binding assays to identify immunogenic epitopes derived from CG. Flow cytometry, immunoblotting, and confocal microscopy were used to characterize the expression and processing of CG in AML patient samples, leukemia stem cells, and normal neutrophils. Cytotoxicity assays determined the susceptibility of AML to CG-specific cytotoxic T lymphocytes (CTL). Dextramer staining and cytokine flow cytometry were conducted to characterize the immune response to CG in patients.

Results: CG was highly expressed and ubiquitinated in AML blasts, and was localized outside granules in compartments that facilitate antigen presentation. We identified five HLA-A*0201 binding nonameric peptides (CG1-CG5) derived from CG, and showed immunogenicity of the highest HLA-A*0201 binding peptide, CG1. We showed killing of primary AML by CG1-CTL, but not normal bone marrow. Blocking HLA-A*0201 abrogated CG1-CTL-mediated cytotoxicity, further confirming HLA-A*0201-dependent killing. Finally, we showed functional CG1-CTLs in peripheral blood from AML patients following allogeneic stem cell transplantation.

Conclusion: CG is aberrantly expressed and processed in AML and is a novel immunotherapeutic target that warrants further development.
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http://dx.doi.org/10.1158/1078-0432.CCR-12-2753DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3537920PMC
January 2013

Quantification of human uridine-diphosphate glucuronosyl transferase 1A isoforms in liver, intestine, and kidney using nanobore liquid chromatography-tandem mass spectrometry.

Anal Chem 2012 Jan 5;84(1):98-105. Epub 2011 Dec 5.

Division of Molecular Pharmaceutics, Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, North Carolina 27599, USA.

Uridine-disphosphate glucuronosyl transferase (UGT) enzymes catalyze the formation of glucuronide conjugates of phase II metabolism. Methods for absolute quantification of UGT1A1 and UGT1A6 were previously established utilizing stable isotope peptide internal standards with liquid chromatography-tandem mass spectrometry (LC-MS/MS). The current method expands upon this by quantifying eight UGT1A isoforms by nanobore high-performance liquid chromatography (HPLC) coupled with a linear ion trap time-of-flight mass spectrometer platform. Recombinant enzyme digests of each of the isoforms were used to determine assay linearity and detection limits. Enzyme expression level in human liver, kidney, and intestinal microsomal protein was determined by extrapolation from spiked stable isotope standards. Intraday and interday variability was <25% for each of the enzyme isoforms. Enzyme expression varied from 3 to 96 pmol/mg protein in liver and intestinal microsomal protein digests. Expression levels of UGT1A7, 1A8, and 1A10 were below detection limits (<1 pmol/mg protein) in human liver microsome (HLMs). In kidney microsomes the expression of UGT1A3 was below detection limits, but levels of UGT1A4, 1A7, 1A9, and 1A10 protein were higher relative to that of liver, suggesting that renal glucuronidation could be a significant factor in renal elimination of glucuronide conjugates. This novel method allows quantification of all nine UGT1A isoforms, many previously not amenable to measurement with traditional methods such as immunologically based assays. Quantitative measurement of proteins involved in drug disposition, such as the UGTs, significantly improves the ability to evaluate and interpret in vitro and in vivo studies in drug development.
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http://dx.doi.org/10.1021/ac201704aDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3259189PMC
January 2012

A new approach to IRMPD using selective ion dissociation in a quadrupole ion trap.

J Am Soc Mass Spectrom 2011 Feb 15;22(2):207-13. Epub 2011 Jan 15.

Department of Chemistry, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-3290, USA.

Infrared multiphoton photodissociation (IRMPD) in a quadrupole ion trap is not selective for a parent ion. Product ions are decreased in abundance by continuous sequential dissociation and may be lost below the low mass cut-off. The IRMPD process is made selective by resonantly exciting trapped ions into an axially offset laser path. Product ions form and collisionally relax out of the laser path to accumulate in the center of the trap. The technique, termed selective broadband (SB) IRMPD, limits sequential dissociation to preserve first generation product ion abundance. The abundances of larger product ions are maximized by completely dissociating the parent ion, but continuous sequential dissociation does not form small product ions below the low mass cut-off associated with conventional IRMPD. Smaller product ions are further increased in abundance in another tandem mass spectrum by performing sequential stages of SB-IRMPD, adjusting the trapping rf amplitude to dissociate larger product ions at the same q(z) range. Thermal assistance is used to perform SB-IRMPD at higher bath gas pressures for increased sensitivity.
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http://dx.doi.org/10.1007/s13361-010-0039-yDOI Listing
February 2011