Further exploration of the collision-induced dissociation of select beta blockers: Acebutolol, atenolol, bisoprolol, carteolol, and labetalol.

Beta blockers are a class of drugs commonly used to treat heart-related diseases; they are also regulated under the World Anti-Doping Agency. Tandem mass spectrometry is often used in the pharmaceutical industry, clinical analysis laboratory, and antidoping laboratory for detection and characterization of drugs and their metabolites. A deeper chemical understanding of dissociation pathways may eventually lead to an improved ability to predict tandem mass spectra of compounds based strictly on their chemical structure (or vice versa), which is especially important for characterization of unknowns such as emerging designer drugs or novel metabolites. In addition to providing insights into dissociation pathways, the use of energy-resolved breakdown curves can produce improved selectivity and lend insights into optimal fragmentation conditions for liquid chromatography-tandem mass spectrometry LC-MS/MS workflows. Here, we perform energy-resolved collision cell and multistage ion trap collision-induced dissociation-mass spectrometry (CID-MS) experiments, along with complementary density functional theory calculations, on five beta blockers (acebutolol, atenolol, bisoprolol, carteolol, and labetalol), to better understand the details of the pathways giving rise to the observed MS/MS patterns. Results from this work are contextualized within previously reported literature on these compounds. New insights into the formation of the characteristic product ion m/z 116 and the pathway leading to characteristic loss of 77 u are highlighted. We also present comparisons of breakdown curves obtained via qToF, quadrupole ion trap, and in-source CID, allowing for differences between the data to be noted and providing a step toward allowing for improved selectivity of breakdown curves to be realized on simple instruments such as single quadrupoles or ion traps.

Infrared multiple photon dissociation (IRMPD) spectroscopy and its potential for the clinical laboratory.

Infrared multiple photon dissociation (IRMPD) spectroscopy is a powerful tool used to probe the vibrational modes-and, by extension, the structure-of an ion within an ion trap mass spectrometer. Compared to traditional FTIR spectroscopy, IRMPD spectroscopy has advantages including its sensitivity and its relative ability to handle complex mixtures. While IRMPD has historically been a technique for fundamental analyses, it is increasingly being applied in a more analytical fashion. Notable recent demonstrations pertinent to the clinical laboratory and adjacent interests include analysis of modified amino acids/residues and carbohydrates, structural elucidation (including isomeric differentiation) of metabolites, identification of novel illicit drugs, and structural studies of various biomolecules and pharmaceuticals. Improvements in analysis time, coupling to commercial instruments, and integration with separations methods are all drivers toward the realization of these analytical applications. Additional improvements in these areas, along with advances in benchtop tunable IR sources and increased cross-discipline collaboration, will continue to drive innovation and widespread adoption. The goal of this tutorial article is to briefly present the fundamentals and instrumentation of IRMPD spectroscopy, as an overview of the utility of this technique for helping to answer questions relevant to clinical analysis, and to highlight limitations to widespread adoption, as well as promising directions in which the field may be heading.

Thermal- and collision-induced dissociation studies of functionalized imidazolium-based ionic liquid cations.

Ionic liquids are now used in applications ranging from chemical synthesis to spacecraft propulsion. With this comes the need to characterize new syntheses, identify environmental contamination, and determine eventual fate in terrestrial and space environments. This work investigates the effects of source conditions, particularly capillary temperature, on the observed mass spectrum and determines the collision-induced dissociation (CID) patterns of imidazolium-based ionic liquid cations as a function of their substituent types. Experiments were carried out on a Thermo LTQ-XL ion-trap mass spectrometer and a Bruker microTOF-Q II mass spectrometer. Dissociation of the imidazolium cations occurred predominantly via substituent losses, except in benzyl-substituted systems, for which the neutral loss of the imidazole was exclusively observed. Several of these dissociation pathways were studied in greater depth using complementary quantum chemical calculations. The nature of the neutral losses from the substituents was found to be highly dependent upon the nature of the substituent, as would be expected, establishing bases for characterization.

A Combined Infrared Ion Spectroscopy and Computational Chemistry Study of Hydroxyproline Isomers.

Hydroxyproline is a common variation of proline, with diverse biological roles. The hydroxylation of proline gives rise to several (natural and/or synthetic) isomeric forms, including both positional isomers and stereoisomers. While mass spectrometry is widely touted as a very selective analytical technique, the identification of closely related isomers often poses a challenge. In these cases, allied technologies become helpful in providing full characterization. Here, infrared multiple photon dissociation (IRMPD) spectroscopy is used to differentiate between three isomers, namely cis-3-hydroxyproline, cis-4-hydroxyproline, and trans-4-hydroxyproline. In contrast to the protonated species which show only minor variations in their IRMPD spectra, lithiated species were found to display significant spectral differences, making their differentiation more straightforward. The conformational origin of these spectral differences was investigated by complementary quantum-chemical calculations.

Electrospray ionization enters the final frontier: Mass spectrometry's role in understanding electrospray thrusters and their plumes.

Electrospray thrusters using ionic liquid (IL)-based propellants are quickly gaining popularity in spacecraft design. Mass spectrometry is especially well-suited to provide important knowledge on the fundamentals of how these systems work and on evaluating their efficiencies and impacts, given that the operating principles of electrospray thrusters closely mimics the mass spectrometry experiment - in both ions are generated by electrospray and then enter a vacuum. Here, electrospray thruster technology and IL-based propellants are briefly introduced. This introduction is then followed by a discussion of mass spectrometry's current contribution to the study of IL-based electrospray thrusters - with a focus on electrospray, dissociation, and spectroscopy studies - and a brief discussion of areas ripe for immediate contributions from the mass spectrometry community.

Mechanistic insights into intramolecular phosphate group transfer during collision induced dissociation of phosphopeptides.

We report on the rearrangement chemistry of model phosphorylated peptides during collision-induced dissociation (CID), where intramolecular phosphate group transfers are observed from donor to acceptor residues. Such "scrambling" could result in inaccurate modification localization, potentially leading to misidentifications. Systematic studies presented herein provide mechanistic insights for the unusually high phosphate group rearrangements presented some time ago by Reid and coworkers (Proteomics 2013, 13 [6], 964-973). It is postulated here that a basic residue like histidine can play a key role in mediating the phosphate group transfer by deprotonating the serine acceptor site. The proposed mechanism is consistent with the observation that fast collisional activation by collision-cell CID and higher-energy collisional dissociation (HCD) can shut down rearrangement chemistry. Additionally, the rearrangement chemistry is highly dependent on the charge state of the peptide, mirroring previous studies that less rearrangement is observed under mobile proton conditions.

Dissociation pathways of protic ionic liquid clusters: Alkylammonium nitrates.

Protic ionic liquids are promising candidates for many applications, including as spacecraft propellants. For both fundamental interest and understanding clustering and dissociation during electrospray-based propulsion, it is useful to explore the dissociation pathways of protic ionic liquid clusters, as well as the factors affecting the relative contributions of each pathway to the observed MS/MS spectra. With that said, most of the published reports on ionic liquid cluster dissociation have focused on aprotic ionic liquids. The purpose of the current work is to explore the dissociation pathways (eg, loss of amine, nitric acid, or ion pair) of alkylammonium nitrates using energy-resolved collision-induced dissociation. Here, it was found that, in general, protic ionic liquids have multiple dissociation pathways-namely, protic ionic liquids can lose their neutralized cation (here, an alkylamine) or neutralized anion (here, nitric acid)-in addition to the ion pair dissociation familiar to aprotic salt and aprotic ionic liquid clusters. In general, increasing the basicity of the cation (here, through increasing the degree of alkylation) decreases the propensity to follow these alternative pathways. Interestingly, increasing the cluster size has a similar effect: as cluster size increases, nitric acid loss decreases. These results will help better model and design protic ionic liquids for electrospray-based spacecraft propulsion and help provide a better understanding for the general behavior of protic ionic liquids versus aprotic ionic liquids within mass spectrometers.

Theoretical and Experimental Insights into the Dissociation of 2-Hydroxyethylhydrazinium Nitrate Clusters Formed via Electrospray.

Ionic liquids are used for myriad applications, including as catalysts, solvents, and propellants. Specifically, 2-hydroxyethylhydrazinium nitrate (HEHN) has been developed as a chemical propellant for space applications. The gas-phase behavior of HEHN ions and clusters is important in understanding its potential as an electrospray thruster propellant. Here, the unimolecular dissociation pathways of two clusters are experimentally observed, and theoretical modeling of hydrogen bonding and dissociation pathways is used to help rationalize those observations. The cation/deprotonated cation cluster [HEH2 - H]+, which is observed from electrospray ionization, is calculated to be considerably more stable than the complementary cation/protonated anion adduct, [HEH + HNO3]+, which is not observed experimentally. Upon collisional activation, a larger cluster [(HEHN)2HEH]+ undergoes dissociation via loss of nitric acid at lower collision energies, as predicted theoretically. At higher collision energies, additional primary and secondary loss pathways open, including deprotonated cation loss, ion-pair loss, and double-nitric-acid loss. Taken together, these experimental and theoretical results contribute to a foundational understanding of the dissociation of protic ionic liquid clusters in the gas phase.

Effects of ESI conditions on kinetic trapping of the solution-phase protonation isomer of p-aminobenzoic acid in the gas phase.

The effects of electrospray ionization (ESI) solvent and source temperature on the relative abundance of the preferred solution-phase (N-protonated; i.e. amine) versus preferred gas-phase (O-protonated; i.e., acid) isomers of p-aminobenzoic acid (PABA) were investigated. When PABA was electrosprayed from protic solvents (i.e., methanol/water), the infrared multiple photon dissociation (IRMPD) spectrum recorded was consistent with that for O-protonation, according to both calculations and previous studies. When aprotic solvent (i.e., acetonitrile) was used, a different spectrum was recorded and was assigned to the N-protonated isomer. As the amine is the preferred protonation site in solution, this suggests that an isomerization takes place under certain conditions. Photodissociation at the diagnostic band for the O-protonated isomer (NH2 stretching mode) was used to quantify the relative contributions of each isomer to ion signal as a function of ESI conditions. For mixtures of methanol and acetonitrile, the relative contribution of the O-protonated gas-phase structure increased as a function of methanol content. Yet, substituting methanol for water resulted in a marked decrease of isomerization to the O-protonated structure. The source temperature (i.e., temperature of a heated desolvation capillary) was found to play a key role in determining the extent of isomerization, with higher temperatures yielding increased presence of gas-phase structures. These results are consistent with a protic bridge mechanism, in which the ESI droplet temperatures, dependent on endothermic desolvation and radiative heating from the capillary, may determine the isomerization yield.

Resuscitation fluid composition affects hepatic inflammation in a murine model of early sepsis.

BACKGROUND: Fluid resuscitation is a crucial therapy for sepsis, and the use of balanced fluids and/or isotonic albumin may improve patient survival. We have previously demonstrated that resuscitation with normal saline results in increased hepatic leukocyte recruitment in a murine model of sepsis. Given that clinical formulations of albumin are in saline, our objectives were to develop a novel balanced electrolyte solution specifically for sepsis and to determine if supplementing this solution with albumin would improve the inflammatory response in sepsis. METHODS: We developed two novel buffered electrolyte solutions that contain different concentrations of acetate and gluconate, named Seplyte L and Seplyte H, and administered these solutions with or without 5% albumin. Normal saline with or without albumin and Ringer's lactate served as controls. Sepsis was induced by cecal ligation and puncture (CLP), and the liver microvasculature was imaged in vivo at 6 h after CLP to quantify leukocyte recruitment. Hepatic cytokine expression and plasma cell-free DNA (cfDNA) concentrations were also measured. RESULTS: Septic mice receiving either Seplyte fluid showed significant reductions in hepatic post-sinusoidal leukocyte rolling and adhesion compared to normal saline. Hepatic cytokine concentrations varied in response to different concentrations of acetate and gluconate in the novel resuscitation fluids but were unaffected by albumin. All Seplyte fluids significantly increased hepatic TNF-α levels at 6 h compared to control fluids. However, Seplyte H exhibited a similar cytokine profile to the control fluids for all other cytokines, whereas mice given Seplyte L had significantly elevated IL-6, IL-10, KC (CXCL1), and MCP-1 (CCL2). Plasma cfDNA was generally increased during sepsis, but resuscitation fluid composition did not significantly affect cfDNA concentrations. CONCLUSIONS: Electrolyte concentrations and buffer constituents of resuscitation fluids can modulate hepatic cytokine production and leukocyte recruitment in septic mice, while the effects of albumin are modest during early sepsis. Therefore, crystalloid fluid choice should be an important consideration for resuscitation in sepsis, and the effects of fluid composition on inflammation in other organ systems should be studied to better understand the physiological impact of this vital sepsis therapy.

H2SO4 and SO3 transfer reactions in a sulfopeptide-basic peptide complex.

We report on the intermolecular transfer of sulfuric acid (H2SO4) and sulfur trioxide (SO3) from an acidic sulfopeptide (sSE) to a basic peptide (R3); this is achieved by subjecting a noncovalent complex of sSE + R3 to collisional activation in a quadrupole ion trap. The product ions resulting from the sulfo-group transfers were characterized by MS(3) experiments. Peak assignments were additionally supported by isotope-labeling and energy-resolved collision induced dissiciation (CID) experiments. The observed reactions and their potential implications for proteomics and post-translational modification discovery experiments are discussed.

Peptide fragmentation products in mass spectrometry probed by infrared spectroscopy.

Vibrational spectroscopy offers detailed insights, by virtue of diagnostic infrared bands, into the chemical structures and moieties which are formed during peptide fragmentation inside mass spectrometers. Over the past few years, IRMPD spectroscopy has led to a greatly improved understanding of the chemistry that takes place during collision-induced dissociation (CID) of protonated peptides. For instance, the rearrangement chemistry of b- and a-type ions, which is relevant in sequence scrambling pathways, has been directly confirmed with the technique. In this chapter, we provide a brief background on peptide fragmentation chemistry, and give an overview of areas where vibrational spectroscopy has been successfully implemented, such as CID of protonated and de-protonated peptides. We also discuss the potential of the technique for elucidating lesser-studied radical dissociation processes, such as electron capture dissociation (ECD), electron transfer dissociation (ETD), and laser photodissociation.

Syndecan-1 in the mouse parietal peritoneum microcirculation in inflammation.

BACKGROUND: The heparan sulfate proteoglycan syndecan-1 (CD138) was shown to regulate inflammatory responses by binding chemokines and cytokines and interacting with adhesion molecules, thereby modulating leukocyte trafficking to tissues. The objectives of this study were to examine the expression of syndecan-1 and its role in leukocyte recruitment and chemokine presentation in the microcirculation underlying the parietal peritoneum. METHODS: Wild-type BALB/c and syndecan-1 null mice were stimulated with an intraperitoneal injection of Staphylococcus aureus LTA, Escherichia coli LPS or TNFα and the microcirculation of the parietal peritoneum was examined by intravital microscopy after 4 hours. Fluorescence confocal microscopy was used to examine syndecan-1 expression in the peritoneal microcirculation using fluorescent antibodies. Blocking antibodies to adhesion molecules were used to examine the role of these molecules in leukocyte-endothelial cell interactions in response to LTA. To determine whether syndecan-1 co-localizes with chemokines in vivo, fluorescent antibodies to syndecan-1 were co-injected intravenously with anti-MIP-2 (CXCL2), anti-KC (CXCL1) or anti-MCP-1 (CCL2). RESULTS AND CONCLUSION: Syndecan-1 was localized to the subendothelial region of peritoneal venules and the mesothelial layer. Leukocyte rolling was significantly decreased with LPS treatment while LTA and TNFα significantly increased leukocyte adhesion compared with saline control. Leukocyte-endothelial cell interactions were not different in syndecan-1 null mice. Antibody blockade of ß2 integrin (CD18), ICAM-1 (CD54) and VCAM-1 (CD106) did not decrease leukocyte adhesion in response to LTA challenge while blockade of P-selectin (CD62P) abrogated leukocyte rolling. Lastly, MIP-2 expression in the peritoneal venules was not dependent on syndecan-1 in vivo. Our data suggest that syndecan-1 is expressed in the parietal peritoneum microvasculature but does not regulate leukocyte recruitment and is not necessary for the presentation of the chemokine MIP-2 in this tissue.

Development of a murine model of early sepsis in diet-induced obesity.

Sepsis, a global health issue, is the most common cause of mortality in the intensive care unit. The aim of this study was to develop a new model of sepsis that investigates the impact of prolonged western diet (WD) induced obesity on the response to early sepsis. Male C57BL/6 mice were fed either a high fat WD or normal chow diet (NCD) for 6, 15, or 27 weeks. Septic obese mice at 15 and 27 weeks had significantly lower levels of lung myeloperoxidase (26.3 ± 3.80 U/mg tissue) compared to age matched ad lib (44.1 ± 2.86 U/mg tissue) and diet restricted (63.2 ± 5.60 U/mg tissue) controls. Low levels of lung inflammation were not associated with changes in hepatic cytokines and oxidative stress levels. Obese mice had significantly (P < 0.0001) larger livers compared to controls. Histological examination of the livers demonstrated that WD fed mice had increased inflammation with pronounced fat infiltration, steatosis, and hepatocyte ballooning. Using this model of prolonged exposure to high fat diet we have data that agree with recent clinical observations suggesting obese individuals are protected from sepsis-induced lung injury. This model will allow us to investigate the links between damage to the hepatic microcirculation, immune response, and lung injury.

Differentiating sulfopeptide and phosphopeptide ions via resonant infrared photodissociation.

The post-translational modifications sulfation and phosphorylation pose special challenges to mass spectral analysis due to their isobaric nature and their lability in the gas phase, as both types of peptides dissociate through similar channels upon collisional activation. Here, we present resonant infrared photodissociation based on diagnostic sulfate and phosphate OH stretches, as a means to differentiate sulfated from phosphorylated peptides within the framework of a mass spectrometry platform. The approach is demonstrated for a number of tyrosine-containing peptides, ranging from dipeptides (YG, pYG, and sYG) over tripeptides (GYR, GpYR, and GsYR), to more biologically relevant enkephalin peptides (YGGFL, pYGGFL, and sYGGFL). In all cases, the diagnostic ranges for sulfate OH stretches are established as 3580-3600 cm(-1) and can thus be distinguished from other characteristic hydrogen stretches, such as carboxylic acid OH, alcohol OH, and phosphate OH stretches.

Alpha-1 acid glycoprotein reduces hepatic leukocyte recruitment in murine models of either early endotoxemia or early sepsis.

OBJECTIVE: To characterize the effect of systemically administered AGP on early leukocyte recruitment in the livers of endotoxemic or septic mice and to determine whether this is influenced by LPS sequestration. METHODS: Endotoxemia was induced in C57Bl/6 mice via intraperitoneal injection of LPS. Sepsis was induced in mice by cecal ligation and perforation. AGP (165 mg/kg) or saline (20 mL/kg) or HAS (200 mg/kg) was administered immediately after surgery or LPS injection and the hepatic microcirculation was examined by intravital microscopy at four hour. RESULTS: Leukocyte adhesion in the PSV was reduced by treatment with AGP in mice subjected to either LPS or CLP protocols compared to either saline or HAS treatment. AGP-treated mice also had significantly higher sinusoidal flow in both models. Pre-incubation of LPS with AGP reduced the ability of LPS to recruit leukocytes to the liver microcirculation. CONCLUSIONS: AGP was more effective in limiting hepatic inflammation and maintaining perfusion than saline or HAS, in both endotoxemic and septic mice. AGP sequestration of LPS may contribute to its anti-inflammatory effects.

Screening for phosphorylated and nonphosphorylated peptides by infrared photodissociation spectroscopy.

We present an infrared laser-based mass spectrometric strategy to differentiate peptides that are phosphorylated (i.e., containing pS, pT, or pY) from those that are nonphosphorylated (i.e., containing S, T, or Y), and those peptides that have none of these moieties (i.e., containing neither pS, pT, pY nor S, T, Y). This is demonstrated for a series of tripeptides and for two larger octapeptides, showing that the diagnostic phosphate OH stretch (indicative for pS, pT, or pY) can be distinguished from the alcohol OH stretch (indicative for S, T, or Y). In addition, the infrared multiple photon dissociation (IRMPD) spectra of multiple peptide analytes are recorded simultaneously in a multiplexed fashion. This is achieved by complexing each peptide precursor with a noncovalently bound 18-crown-6 ether, which is detached upon resonant infrared photon absorption.

Optical imaging in tissue with X-ray excited luminescent sensors.

We report a high-spatial resolution imaging technique to measure optical absorption and detect chemical and physical changes on surfaces embedded in thick tissue. Developing sensors to measure chemical concentrations on implanted surfaces through tissue is an important challenge for analytical chemistry and biomedical imaging. Tissue scattering dramatically reduces the resolution of optical imaging. In contrast, X-rays provide high spatial resolution imaging through tissue but do not measure chemical concentrations. We describe a hybrid technique which uses a scanning X-ray beam to irradiate Gd(2)O(2)S scintillators and detect the resulting visible luminescence through the tissue. The amount of light collected is modulated by optical absorption in close proximity to the luminescence source. By scanning the X-ray beam, and measuring total amount of light collected, one can measure the local absorption near scintillators at a resolution limited by the width of luminescence source (i.e. the width of the X-ray excitation beam). For proof of principle, a rectangular 1.7 mm scanning X-ray beam was used to excite a single layer of 8 µm Gd(2)O(2)S particles, and detect the absorption of 5 nm thick silver island film through 10 mm of pork. Lifetime and spectroscopic measurements, as well changing the refractive index of the surroundings indicate that the silver reduces the optical signal through attenuated total internal reflection. The technique was used to image the dissolution of regions of the silver island film which were exposed to 1 mM of H(2)O(2) through 1 cm of pork tissue.

High-resolution chemical imaging through tissue with an X-ray scintillator sensor.

We describe a novel method for high-resolution chemical imaging on a surface embedded in tissue. The sensor surface consists of an X-ray scintillator film coated in a thin film loaded with chemical indicator dye. A narrow scanning X-ray beam is used to excite luminescence from X-ray scintillators located within the beam. This luminescence passes through the indicator film, and the spectrum is analyzed to measure chemical concentrations at that location. A pH sensor is demonstrated with a dynamic range between pH 6-9 and noise level of 0.05 pH units using methyl-red dyed pH paper. The location of the interface between two types of scintillator films is obtained with 0.30 mm spatial resolution even though the images are highly blurred by 10 mm of chicken breast. This work has important applications for detecting pH changes on surfaces of implanted medical devices.

Innate immunity of the liver microcirculation.

The liver is a complex organ with a unique microcirculation and both synthetic and immune functions. Innate immune responses have been studied in response to single inflammatory mediators and several clinically relevant models of infection and injury. While standard histological techniques have been used in many models, the liver microcirculation is also amenable to in vivo examination using epifluorescent, confocal and transillumination intravital microscopy. These techniques have begun to clarify not only the molecular mechanisms but also the specific cell populations involved in the liver inflammation. In this review, we discuss the cells and mediators involved in hepatic innate immunity in simple and complex models of injury and infection, and present the view that the liver microcirculation utilizes non-classical pathways for leukocyte recruitment.