The rapid and on-line study of aerosols and their properties is technically demanding due to their small size (<10 μm diameter) and the resultant required scale of any such measurements. Most such techniques require the use of lasers (e.g., phase Doppler anemometry), condensation growth, or other complex hardware. To this end we introduce analysis of liquid particles in aerosols via charge-induction amperometry (ALPACA), an extremely simple potentiostat-based technique capable of on-line, rapid measurement of the aggregate charge of aerosol particles. This technique demonstrates high signal-to-noise responses, is not subject to chemical noise, and has the potential for significant future miniaturization. This technique is applied in this work for the detection of charges on electrosprayed droplets. The mechanism of detection of the technique is discussed using both amperometry and open circuit potential (OCP) to measure droplet charge properties. ALPACA represents a significant advancement toward simple, inexpensive aerosol charge detection.

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.

Electrospray ionization (ESI) is a powerful ionization technique that can generate charged solvent droplets and bare analyte ions from sample solutions. Despite seeing extensive use in mass spectrometry due in part to the low internal energy deposited into the ions formed during ionization, some unknowns persist regarding the exact dynamics of droplet breakup and molecule behavior during spray, and research is still underway regarding how various types of molecules acquire charge during the ESI process. Previously, the authors introduced a novel aerosol measurement technique, particle-into-liquid sampling for nanoliter electrochemical reactions (PILSNER). The current work introduces a new method utilizing PILSNER for the examination of the particles generated during ESI using simple analysis techniques with a commercially available potentiostat. This technique is applied in this work for the detection of charges on electrosprayed droplets, including the estimation of the number of charges on individual ESI droplets using a fluorescent proxy. This technique provides an additional tool for the exploration of the complex process of droplet generation and ion liberation during ESI.

The document contains 4 supplemental tables: 1. RT-PCR Primers for TCRbeta sequencing 2. HPLC-MS identified peptide sequences 3. The amino acid sequences of recurrent TCRbeta clonotypes 4. The distribution of the CDR3 amino acid length in the analyzed patient. There are 2 supplementary figures: 1. Tetramer flow cytometry showing the generation of an oligoclonal T-cell population with a UNC-CDK4-1 high affinity T-cell population. 2. More detailed information regarding the tetramer gating strategy employed.

<p>PDF file - 706k, Figure S1. CG is ubiquitinated in AML. Figure S2. CG1 is a naturally processed epitope that is eluted from leukemia cell surface. Figure S3. CG expression by each of the AML samples used in the cytotoxicity assays. Figure S4. Gating strategy used to determine CG1-CTL frequency in AML patient blood. Supplementary Table S1. Characteristics of cathepsin G-derived peptides with highest binding affinity to HLA-A*0201 Supplementary Table S2. AML samples used in determining cathepsin G expression</p>

Lipid droplets (LDs) are intracellular storage vesicles composed of a neutral lipid core surrounded by a glycerophospholipid membrane. LD accumulation is associated with different stages of cancer progression and stress responses resulting from chemotherapy. In previous work, a novel dual nano-electrospray ionization source and data-dependent acquisition method for measuring the relative abundances of lipid species between two extracts were described and validated. Here, this same source and method were used to determine if oxaliplatin-sensitive and resistant cells undergo similar lipid profile changes, with the goal of identifying potential signatures that could predict the effectiveness of an oxaliplatin-containing treatment. Oxaliplatin is commonly used in the treatment of colorectal cancer. When compared to a no-drug control, oxaliplatin dosing caused significant increases in triglyceride (TG) and cholesterol ester (CE) species. These increases were more pronounced in the oxaliplatin-sensitive cells than in oxaliplatin-resistant cells. The increased neutral lipid abundance correlated with LD formation, as confirmed by confocal micrographs of Nile Red-stained cells. Untargeted proteomic analyses also support LD formation after oxaliplatin treatment, with an increased abundance of LD-associated proteins in both the sensitive and resistant cells.

Abstract Motivation Splice variant neoantigens are a potential source of tumor-specific antigen (TSA) that are shared between patients in a variety of cancers, including acute myeloid leukemia. Current tools for genomic prediction of splice variant neoantigens demonstrate promise. However, many tools have not been well validated with simulated and/or wet lab approaches, with no studies published that have presented a targeted immunopeptidome mass spectrometry approach designed specifically for identification of predicted splice variant neoantigens. Results In this study, we describe NeoSplice, a novel computational method for splice variant neoantigen prediction based on (i) prediction of tumor-specific k-mers from RNA-seq data, (ii) alignment of differentially expressed k-mers to the splice graph and (iii) inference of the variant transcript with MHC binding prediction. NeoSplice demonstrates high sensitivity and precision (>80% on average across all splice variant classes) through in silico simulated RNA-seq data. Through mass spectrometry analysis of the immunopeptidome of the K562.A2 cell line compared against a synthetic peptide reference of predicted splice variant neoantigens, we validated 4 of 37 predicted antigens corresponding to 3 of 17 unique splice junctions. Lastly, we provide a comparison of NeoSplice against other splice variant prediction tools described in the literature. NeoSplice provides a well-validated platform for prediction of TSA vaccine targets for future cancer antigen vaccine studies to evaluate the clinical efficacy of splice variant neoantigens. Availability and implementation https://github.com/Benjamin-Vincent-Lab/NeoSplice Supplementary information Supplementary data are available at Bioinformatics Advances online.

Particle-into-liquid sampling (PILS) has enabled robust quantification of analytes of interest in aerosol particles. In PILS, the limit of detection is limited by the factor of particle dilution into the liquid sampling volume. Thus, much lower limits of detection can be achieved by decreasing the sampling volume and increasing the surface area-to-volume ratio of the collection substrate. Unfortunately, few analytical techniques can realize this miniaturization. Here, we use an ultramicroelectrode in a microliter or smaller sampling volume to detect redox active species in aerosols to develop the technique of Particle-into-Liquid Sampling for Nanoliter Electrochemical Reactions (PILSNER). As a proof-of-concept to validate this technique, we demonstrate the detection of K4Fe(CN)6 in aerosol particles (diameter ∼0.1-2 μm) and quantify the electrochemical response. To further explore the utility of the method to detect environmentally relevant redox molecules, we show PILSNER can detect 1 ng/m3 airborne Pb in aerosols. We also demonstrate the feasibility of detecting perfluorooctanesulfonate (PFOS), a persistent environmental contaminant, using this technique. PILSNER is shown to represent a significant advancement toward simple and effective detection of a variety of emerging contaminants with an easily miniaturizable and tunable electroanalytical platform.

Paper-based cultures are an emerging platform for preparing three-dimensional (3D) tissue- and tumor-like structures. The ability to stack individual sheets of cell-containing paper affords a modular means of assembling structures with defined cellular compositions and microenvironments. These layered stacks are easily separated at the end of an experiment, providing spatially resolved populations of live cells for further analysis. Here we describe a workflow in which cell viability, drug penetration, and drug metabolism are quantified in a spatially resolved manner. Specifically, we mapped the distribution of the drug irinotecan and its bioactive metabolite SN38 in a colorectal cancer cell-containing stacked structure with liquid chromatography-mass spectrometry (LC-MS). This paper provides the first example of a 3D culture platform that quantifies viability and drug metabolism in a spatially resolved manner. Our data show that cells at the bottom of the stack are more drug-resistant than layers in contact with the culture medium, similar to cells in the nutrient-poor center of a proliferating tumor being more drug-resistant than the rapidly dividing cells at its periphery. The powerful combination of quantitative viability and drug metabolism measurements will enable future studies to determine the exact mechanism(s) of drug resistance in different regions of a tumor.

The COVID-19 pandemic, caused by the virus SARS-cov-2, has been serious global health crisis. A common symptom in patients with severe cases of COVID-19 is dangerously low blood oxygen levels. Treatment of low blood oxygen requires supplementary oxygen delivery using external means, including noninvasive nasal cannulae, rebreathing masks, and non- invasive ventilators (NIV devices), and in severe cases anesthesia and invasive intubation with ventilation. Though effective, invasive intubation is dangerous due in part to the application of anesthetic, and intubated patient outcomes tend to be poor. NIV devices would thus be preferable when patients exhibit only modest symptoms. However, unknowns still exist surrounding whether or not NIV oxygen delivery methods produce potentially infectious virus-containing aerosols. Invasive ventilation involving intubation is a completely closed system and is treated as aerosol-free and less likely to spread infection. Thus, the choice of whether to prioritize the health of patients or healthcare workers has been a dilemma. Current work has explored the possibility that dangerous amounts of aerosol are produced by noninvasive oxygen delivery devices via a mannequin simulating a patient by detecting aerosol under various parameters. Preliminary data shows the efficacy of this mannequin model as well as evidence that noninvasive ventilation may produce more aerosol than nasal cannulae or a control.

An aerosol quadrupole ion trap mass spectrometer is reported that is sensitive, has unique capabilities to perform chemical ionization, is operated in real-time, and is able to perform tandem mass spectrometry. The instrument samples particles with an aerodynamic lens and volatilizes them within the heated ion trap electrode assembly. Analyte molecules are ionized within the ion trap by proton transfer from reagent ions, and resultant fragmentation is reduced compared to vacuum UV photoionization. Particle concentrations can be detected linearly over two orders of magnitude and as low as 5 μg/m3. To demonstrate the real-time analysis capability of the instrument, secondary organic aerosol particles were produced by reaction of 100 ppb α-pinene and 200 ppb ozone in an aerosol bag and observed in real-time to monitor the progress of the reaction. Pinic acid and pinonic acid are two of the many components of the secondary aerosol mixture that form and gradually decrease in concentration. Individual concentrations are calculated using pinic acid as an internal standard and vary from 4-36 ppb. The identities of analyte ions from both compounds are confirmed by tandem mass spectrometry in real-time.

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 ex vivo and in vivo 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.

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]2+ 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]2+ 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.

Introduction: The use of flavors in electronic cigarettes appeals to adults and never-smoking youth. Consumption has rapidly increased over the last decade, and in the U.S. market alone, there are over 8000 unique flavors. The U.S. Food and Drug Administration (FDA) has begun to regulate e-liquids, but many have not been tested, and their impact, both at the cellular level, and on human health remains unclear. Methods: We tested e-liquids on the human cell line HEK293T and measured toxicity, mitochondria! membrane potential (Delta Psi(m)), reactive oxygen species production (ROS), and cellular membrane potential (V-m) using high-throughput screening (HTS) approaches. Our HTS efforts included single-dose and 16-point dose-response curves, which allowed testing of AO commercially available e-liquids in parallel to provide a rapid assessment of cellular effects as a proof of concept for a fast, preliminary toxicity method. We also investigated the chemical composition of the flavors via gas chromatography-mass spectrometry. Results: We found that e-liquids caused a decrease in Delta Psi(m) and V-m and an increase in ROS production and toxicity in a dose-dependent fashion. In addition, the presence of five specific chemical components: vanillin, benzyl alcohol, acetoin, cinnamaldehyde, and methyl-cyclopentenolone, but not nicotine, were linked with the changes observed in the cellular traits studied. Conclusion: Our data suggest that Delta Psi(m), ROS, V-m, and toxicity may be indicative of the extent of cell death upon e-liquid exposure. Further research on the effect of flavors should be prioritized to help policy makers such as the FDA to regulate e-liquid composition.

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 Ca2+ is a ubiquitous second messenger that regulates cell growth, we characterized the effects of e-liquids on cellular Ca2+ homeostasis. To better understand the extent of this effect, we screened e-liquids for their ability to alter cytosolic Ca2+ levels and found that 42 of 100 flavored e-liquids elicited a cellular Ca2+ response. Banana Pudding (BP) e-liquid, a representative e-liquid from this group, caused phospholipase C activation, endoplasmic reticulum (ER) Ca2+ release, store-operated Ca2+ entry (SOCE), and protein kinase C (PKCα) phosphorylation. However, longer exposures to BP e-liquid depleted ER Ca2+ stores and inhibited SOCE, suggesting that this e-liquid may alter Ca2+ homeostasis by short- and long-term mechanisms. Since dysregulation of Ca2+ signaling can cause chronic inflammation, ER stress, and abnormal cell growth, flavored e-cigarette products that can elicit cell Ca2+ responses should be further screened for potential toxicity.

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.

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.

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.