Jason W. Flora

Solution composition and thermal denaturation for the production of single-stranded PCR amplicons: piperidine-induced destabilization of the DNA duplex?

Strategies to produce single-stranded PCR amplicons for detection by electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR-MS) were investigated using modified electrospray solutions and by thermally denaturing the duplex structures with a resistively heated electrospray ionization source. A synthetic 20-mer oligonucleotide annealed to its complementary strand was used as a model system for initial experiments. Electrospray solutions were altered by varying the relative proportion of aqueous phase in efforts to induce destabilization of the double helix. When the electrospray solution contains a 25% aqueous content, the 20-mer oligonucleotide is detected in its double-stranded form. Increasing the proportion of aqueous phase in the electrospray solution to 60% destabilized the double helix, resulting in the detection of only single-stranded species. This strategy was extended to an 82-bp polymerase chain reaction (PCR) product derived from the human tyrosine hydroxylase gene (HUMTH01). In efforts to destabilize the 82-bp PCR product, electrospray solutions reaching 70% aqueous content were necessary to promote the detection of only single-stranded amplicons. Implementation of the resistively heated transfer line and an electrospray solution in which the oligonucleotide is on the threshold of duplex stability allowed for double-stranded and single-stranded species to be generated from the same ESI solutions at both ambient and elevated transfer line temperatures, respectively, without disruption of the electrospray process. The volatile base piperidine, present at 20 mM concentrations in the electrospray solution, was found to play a critical role in the formation of single-stranded species at the higher aqueous percentages and a duplex destabilization mechanism has been proposed.

Characterization of potential impurities and degradation products in electronic cigarette formulations and aerosols

E-cigarettes are gaining popularity in the U.S. as well as in other global markets. Currently, limited published analytical data characterizing e-cigarette formulations (e-liquids) and aerosols exist. While FDA has not published a harmful and potentially harmful constituent (HPHC) list for e-cigarettes, the HPHC list for currently regulated tobacco products may be useful to analytically characterize e-cigarette aerosols. For example, most e-cigarette formulations contain propylene glycol and glycerin, which may produce aldehydes when heated. In addition, nicotine-related chemicals have been previously reported as potential e-cigarette formulation impurities. This study determined e-liquid formulation impurities and potentially harmful chemicals in aerosols of select commercial MarkTen(®) e-cigarettes manufactured by NuMark LLC. The potential hazard of the identified formulation impurities and aerosol chemicals was also estimated. E-cigarettes were machine puffed (4-s duration, 55-mL volume, 30-s intervals) to battery exhaustion to maximize aerosol collection. Aerosols analyzed for carbonyls were collected in 20-puff increments to account for analyte instability. Tobacco specific nitrosamines were measured at levels observed in pharmaceutical grade nicotine. Nicotine-related impurities in the e-cigarette formulations were below the identification and qualification thresholds proposed in ICH Guideline Q3B(R2). Levels of potentially harmful chemicals detected in the aerosols were determined to be below published occupational exposure limits.

Carbon-centered radicals in cigarette smoke: acyl and alkylaminocarbonyl radicals.

The widely accepted mechanism of formation for carbon-centered radicals in the gas-phase cigarette smoke involves reactions of NO(2) and alkadienes. However, specific examples of such radicals have never been isolated from fresh cigarette smoke or their structure determined. We have identified two previously unrecognized classes of carbon-centered radicals, alkylaminocarbonyl and acyl radicals, that are unrelated to radicals that form by NO(x) chemistry. The combined abundance of these mainstream smoke radicals is significantly higher than the alkyl radicals previously quantified by electron paramagnetic resonance (EPR) solution spin-trapping methods. The new radicals were trapped directly from smoke with either 3-amino-proxyl (3AP) or 3-cyano-proxyl radical on a solid support and identified by combination of chemical synthesis, deuterium labeling, high-resolution mass spectrometry, nuclear magnetic resonance (NMR) spectroscopy, and ab initio quantum mechanical calculations. 3AP-R adducts were quantified both by high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) and by high-performance liquid chromatography with fluorescence detection (HPLC/FLD). Seven acyl and 11 alkylaminocarbonyl radicals were identified in the whole smoke of cigarettes made from single tobacco varieties and blended tobacco research cigarettes. The overall yield of these radicals was measured to be 168-245 nmol/cigarette from machine-smoked cigarettes under Federal Trade Commission (FTC) conditions. The yield was significantly reduced when the gas-phase smoke was separated from whole smoke by filtration through a 0.1 microm Cambridge filter pad or upon aging whole smoke in an inert tube.

Method for the Determination of Carbonyl Compounds in E-Cigarette Aerosols

Low levels of thermal degradation products such as carbonyls (formaldehyde, acetaldehyde, acrolein, crotonaldehyde) have been reported in e-cigarette aerosols. The collection and analysis of e-cigarette aerosol carbonyls are often adapted from methods developed for tobacco cigarette smoke. These methodologies are often not sensitive enough to detect low carbonyl levels in e-cigarette aerosols. One objective of this work was to develop and validate a rapid, selective and sensitive ultra-performance liquid chromatography with mass spectrometry method optimized for analysis of carbonyls in e-cigarette aerosols. Aerosols were trapped in 20-puff collections, 4-s durations, 55-mL volumes, 30-s intervals, square wave puff profiles. Collection apparatus involved a linear smoking machine with Cambridge filter pad followed by a glass impinger containing acidified 2,4-dinitrophenylhydrazine. This method showed limits of quantitation and detection of 0.016 and 0.003 µg puff−1, respectively, and run time of 4 min. Six e-cigarettes were evaluated (five devices each). All contained measurable levels of carbonyls. Levels were mostly well below those in conventional cigarettes. However, for some e-cigarettes, formaldehyde levels were above those for tobacco cigarettes (highest at 14.1 µg puff−1). Temperatures related to carbonyl yields in e-cigarette aerosols were explored to better understand carbonyl formation: formation of formaldehyde is low at temperatures below 350°C.

Proteomic biomarkers in plasma that differentiate rapid and slow decline in lung function in adult cigarette smokers with chronic obstructive pulmonary disease (COPD)

Chronic obstructive pulmonary disease (COPD) is the fourth leading cause of morbidity and mortality in the United States and cigarette smoking is a primary determinant of the disease. COPD is characterized by chronic airflow limitation as measured by the forced expiratory volume in one second (FEV1). In this study, the plasma proteomes of 38 middle-aged or older adult smokers with mild to moderate COPD, with FEV1 decline characterized as either rapid (RPD, n = 20) or slow or absent (SLW, n = 18), were interrogated using a comprehensive high-throughput proteomic approach, the accurate mass and time (AMT) tag technology. This technology is based upon a putative mass and time tag database (PMT), high-resolution LC separations and high mass accuracy measurements using FT-ICR MS with a 9.4-T magnetic field. The peptide and protein data were analyzed using three statistical approaches to address ambiguities related to the high proportion of missing data inherent to proteomic analysis. The RPD and SLW groups were differentiated by 55 peptides which mapped to 33 unique proteins. Twelve of the proteins have known roles in the complement or coagulation cascade and, despite an inability to adjust for some factors known to affect lung function decline, suggest potential mechanistic biomarkers associated with the rate of lung function decline in COPD. Whether these proteins are the cause or result of accelerated decline will require further research.

Identification of in vitro differential cell secretions due to cigarette smoke condensate exposure using nanoflow capillary liquid chromatography and high-resolution mass spectrometry

Secreted proteins, the secretome, can be isolated from biological fluids (e.g., blood) and are often responsible for the regulation of biological processes such as cell signaling, growth, and apoptosis. The identification of secreted proteins can lead to an understanding of disease mechanisms and they can serve as early candidate biomarkers of disease and exposure. However, it is time-consuming and costly to conduct in vivo interrogations of the human secretome. The purpose of this article is to provide a detailed description of a rapid in vitro technique for the analysis of differential protein secretion due to exposure to smoking-machine-generated cigarette smoke (CS) condensate (total particulate matter, TPM). Endothelial cells were exposed to CS-TPM, the supernatant was collected, and the secretome was elucidated by nano liquid chromatography coupled with high-resolution mass spectrometry. A total of 1,677 unique peptides were identified in the cell culture supernatants. Several proteins were differentially expressed following CS-TPM exposure that relate to several biological processes, such as metabolism, development, communication, response to stimulus, and response to stress.

Comprehensive nomenclature for the fragment ions produced from collisional activation of peptide nucleic acids

A relatively new class of DNA mimics, peptide nucleic acids (PNA’s), have generated increasing interest and importance over the last few years. These molecules have a neutral ‘peptide-like’ backbone granting them many advantageous properties which lead to their potential use as antisense and antigene therapeutics. Due to the promising properties possessed by PNA’s, there is a demand for the development of analytical methods for characterization of these molecules. We have been investigating the gas-phase fragmentation pathways of singly and multiply charged PNA’s using electrospray ionization in conjunction with Fourier transform ion cyclotron resonance mass spectrometry. Fragmentations induced by sustained off-resonance irradiation and nozzle–skimmer dissociation have revealed water loss, cleavages of the methylene carbonyl linker (to which the nucleobases are attached), fragmentation along the PNA backbone, and the elimination of single nucleobases. It is becoming increasingly evident that multi-stage MS is especially suited to structural characterization of large bio-molecules such as PNA’s, and herein we propose a comprehensive nomenclature for the product ions produced upon collisional activation of PNA’s, which is based upon extensive experimental studies.

1H NMR and electrospray mass spectrometry of the mono-ionized bis(2,2'-bis(4,5-dimethylimidazole)chloronitrosylruthenium(II) complex ([Ru(NO)Cl(LH2)2]+, LH2 = 2,2'-bis(4,5-dimethylimidazole)

The reaction of RuCl3(NO)(H2O)2 with 2 equiv. of LH2=2,2′-bis(4,5-dimethylimidazole) in refluxing ethanol generates cis-[Ru(NO)Cl(LH2)2]2+ (1), analogous in structure to cis-[Ru(NO)Cl(bpy)2]2+, as the chloride salt. In 1, the methyl groups are differentiated into eight different 1H NMR magnetic environments. Substitution of 4,5-dimethyl-2,2′-biimidazole (L′H2), an 11.1% impurity in commercial LH2, occurred randomly with the undecorated ring diminishing the intensity of all eight resonances equally. The methyl group of the “out-of-plane” ring that hangs over the π orbitals of the NO+ ligand (CH3(6)) is shifted most upfield from 2.19 ppm of free LH2 and is assigned to the 1.23 ppm resonance. With the aid of 2-D NMR methods, we assign the following shifts. The ring donor opposite the {Ru(NO)}3+, CH3(7), should experience the greatest withdrawing influence, and is assigned as the origin of the 2.50 ppm resonance. Its ring partner, CH3(5), placed below another ring current, is assigned to the resonance at 2.13 ppm. 2-D NMR methods support the assignments of 1.39 ppm to CH3(1) and 2.09 ppm to CH3(2), the “in-plane-near” CH3 groups; 2.36 and 2.34 ppm to “in-plane-remote” CH3(3) and CH3(4); a 2.47 ppm shift is assigned to the remaining CH3(8). Because of the presence of the impurity L′H2 which has a substitution rate advantage (ca. 2.14-fold) due to a lower steric barrier for the unmethylated ring, the isolated product contained 62.4% (1a) [Ru(NO)Cl(LH2)2]Cl2, 31.9% (1b) [Ru(NO)Cl(LH2)(L′H2)]Cl2, and 5.7% (1c) [Ru(NO)Cl(L′H2)2]Cl2. The ESI MS spectrum exhibits parent 1+ ions (2a–c) in which one of the imidazole rings has been deprotonated. Thus, eight-line patterns for RuCl-containing fragments appear for m/z with z=1+ centered about 546 (2a), 518 (2b) and 490 (2c) in the intensity ratios of 1.000:0.511:0.091, respectively. The atomic composition for 2a was shown to be RuC20H27N9OCl (m/z=540.109049) by checking the “540” line of the isotopic bundle around m/z=546. The atomic composition of 2b was established from the “512” mass as RuC18H23N9OCl (m/z=512.077748) from the isotopic bundle around m/z=518. 2a–c undergo the loss of LH2 or L′H2 with low efficiency, but few other fragmentations were observed. Notably, the loss of NO or HCl are absent. 2a–c have good π-donating imidazole and imidazolato functionalities which suppress NO loss. IR laser loss experiments on 2a in the mass spectral cavity were unable to identify a frequency value that selectively dissociates NO. Rather, complete fragmentation of the complexes 2a–c occurred at energies sufficient to induce any ligand dissociation; the parent ions being very robust. The 1H NMR data are supported by an MMFF94 energy-minimized structure for 1 and its theoretical trans isomer.

High-resolution mass spectrometry proteomics for the identification of candidate plasma protein biomarkers for chronic obstructive pulmonary disease

Although cigarette smoking is recognized as the most important cause of chronic obstructive pulmonary disease (COPD), the pathophysiological mechanisms underlying the lung function decline are not well understood. Using off-line strong cation exchange fractionation with RP-LC-ESI-MS/MS and robust database searching, 1758 tryptic peptides were identified in plasma samples from cigarette smokers. Using two statistical approaches, 30 peptides were identified to be associated with the annualized rate of lung function decline over 5 years among smokers with COPD characterized as having rapid (n = 18) or slow (n = 18) decline and 18 smokers without COPD. The identified peptides belong to proteins that are involved in the complement or coagulation systems or have antiprotease or metabolic functions. This research demonstrates the utility of proteomic profiling to improve the understanding of molecular mechanisms involved in cigarette smoking-related COPD by identifying plasma proteins that correlate with decline in lung function.

Characterization of the mouse bronchoalveolar lavage proteome by micro-capillary LC-FTICR mass spectrometry

Bronchoalveolar lavage fluid (BALF) contains proteins derived from various pulmonary cell types, secretions and blood. As the characterization of the BALF proteome will be instrumental in establishing potential biomarkers of pathophysiology in the lungs, the objective of this study was to contribute to the comprehensive collection of Mus musculus BALF proteins using high resolution and highly sensitive micro-capillary liquid chromatography (microLC) combined with state-of-the-art high resolution mass spectrometry (MS). BALF was collected from ICR and C57BL/6 male mice exposed to nose-only inhalation to either air or cigarette smoke. The tandem mass spectra were analyzed by SEQUEST for peptide identifications with the subsequent application of accurate mass and time tags resulting in the identification of 1797 peptides with high confidence by high resolution MS. These peptides covered 959 individual proteins constituting the largest collection of BALF proteins to date. High throughput monitoring profiles of this extensive collection of BALF proteins will facilitate the discovery and validation of biomarkers that would elucidate pathogenic or adaptive responses of the lungs upon toxic insults.