Complement membrane attack complex is an immunometabolic regulator of NLRP3 activation and IL-18 secretion in human macrophages.

The complement system is an ancient and critical part of innate immunity. Recent studies have highlighted novel roles of complement beyond lysis of invading pathogens with implications in regulating the innate immune response, as well as contributing to metabolic reprogramming of T-cells, synoviocytes as well as cells in the CNS. These findings hint that complement can be an immunometabolic regulator, but whether this is also the case for the terminal step of the complement pathway, the membrane attack complex (MAC) is not clear. In this study we focused on determining whether MAC is an immunometabolic regulator of the innate immune response in human monocyte-derived macrophages. Here, we uncover previously uncharacterized metabolic changes and mitochondrial dysfunction occurring downstream of MAC deposition. These alterations in glycolytic flux and mitochondrial morphology and function mediate NLRP3 inflammasome activation, pro-inflammatory cytokine release and gasdermin D formation. Together, these data elucidate a novel signalling cascade, with metabolic alterations at its center, in MAC-stimulated human macrophages that drives an inflammatory consequence in an immunologically relevant cell type.

Acoustic Mist Ionization-Mass Spectrometry: A Comparison to Conventional High-Throughput Screening and Compound Profiling Platforms.

Drug discovery usually begins with a high-throughput screen (HTS) of thousands to millions of molecules to identify starting points for medicinal chemistry. Conventional HTS platforms require expensive reagents and typically have complex assay formats. HTS platforms based on radioactivity are expensive, both in terms of reagent cost and disposal. Furthermore, nonspecific interferences common to these technologies result in an extensive attrition of hits during validation experiments. Mass spectrometry (MS) is a highly selective, label-free technology that can quantify multiple analytes in a single experiment. However, most commercial MS platforms typically involve a separation or cleanup prior to analysis and are too slow for large-scale screening campaigns. Recently, an MS platform (AMI-MS) was introduced that uses acoustically generated droplets to deliver analyte molecules directly from microtiter plates into the mass spectrometer at subsecond per well sampling rates. Here, we demonstrate the application of AMI-MS by developing an HTS-compatible assay that measures the inhibition of histone acetyltransferase activity. Real-time kinetic measurements from a single well were used to determine enzyme Km and Vmax values. We compare the AMI-MS readout with conventional platforms in single-shot screening and multipoint profiling modes. The AMI-MS assay identified 86% of hits previously identified, with a pIC50 ≥ 5.0, in a scintillation proximity assay (SPA) HTS at a lower hit rate and with a significantly reduced cost per well compared to the SPA-based readout. Furthermore, pIC50s, as measured by AMI-MS, showed a good correlation with values generated by RapidFire-MS. AMI-MS has the potential to provide significant improvements to high-throughput bioassays.

Gas phase ion chemistry of an ion mobility spectrometry based explosive trace detector elucidated by tandem mass spectrometry.

The gas phase ion chemistry for an ion mobility spectrometer (IMS) based explosive detector has been elucidated using tandem mass spectrometry. The IMS system, which is operated with hexachloroethane and isobutyramide reagent gases and an ion shutter type gating scheme, is connected to the atmospheric pressure interface of a triple quadrupole mass spectrometer (MS/MS). Product ion masses, daughter ion masses, and reduced mobility values for a collection of nitro, nitrate, and peroxide explosives measured with the IMS/MS/MS instrument are reported. The mass and mobility data together with targeted isotopic labeling experiments and information about sample composition and reaction environment are leveraged to propose molecular formulas, structures, and ionization pathways for the various product ions. The major product ions are identified as [DNT-H](-) for DNT, [TNT-H](-) for TNT, [RDX+Cl](-) and [RDX+NO2](-) for RDX, [HMX+Cl](-) and [HMX+NO2](-) for HMX, [NO3](-) for EGDN, [NG+Cl](-) and [NG+NO3](-) for NG, [PETN+Cl](-) and [PETN+NO3](-) for PETN, [HNO3+NO3](-) for NH4NO3, [NO2](-) for DMNB, [HMTD-NC3H6O3+H+Cl](-) and [HMTD+H-CH2O-H2O2](+) for HMTD, and [(CH3)3CO2](+) for TATP. In general, the product ions identified for the IMS system studied here are consistent with the product ions reported previously for an ion trap mobility spectrometer (ITMS) based explosive trace detector, which is operated with dichloromethane and ammonia reagent gases and an ion trap type gating scheme. Differences between the explosive trace detectors include the [NG+Cl](-) and [PETN+Cl](-) product ions being major ions in the IMS system compared to minor ions in the ITMS system as well as the major product ion for TATP being [(CH3)3CO2](+) for the IMS system and [(CH3)2CNH2](+) for the ITMS system.

Characterization of TATP gas phase product ion chemistry via isotope labeling experiments using ion mobility spectrometry interfaced with a triple quadrupole mass spectrometer.

Identification of the fragment ion species associated with the ion reaction mechanism of triacetone triperoxide (TATP), a homemade peroxide-based explosive, is presented. Ion mobility spectrometry (IMS) has proven to be a key analytical technique in the detection of trace explosive material. Unfortunately, IMS alone does not provide chemical identification of the ions detected; therefore, it is unknown what ion species are actually formed and separated by the IMS. In IMS, ions are primarily characterized by their drift time, which is dependent on the ion׳s mass and molecular cross-section; thus, IMS as a standalone technique does not provide structural signatures, which is in sharp contrast to the chemical and molecular information that is generally obtained from other customary analytical techniques, such as NMR, Raman and IR spectroscopy and mass spectrometry. To help study the ion chemistry that gives rise to the peaks observed in IMS, the hardware of two different commercial IMS instruments has been directly coupled to triple quadrupole (QQQ) mass spectrometers, in order to ascertain each ion׳s corresponding mass/charge (m/z) ratios with different dopants at two temperatures. Isotope labeling was then used to help identify and confirm the molecular identity of the explosive fragment and adduct ions of TATP. The m/z values and isotope labeling experiments were used to help propose probable molecular formulas for the ion fragments. In this report, the fragment and adduct ions m/z 58 and 240 of TATP have been confirmed to be [C3H6NH·H](+) and [TATP·NH4](+), respectively; while the fragment ions m/z 73 and 89 of TATP are identified as having the molecular formulas [C4H9NH2](+) and [C4H9O2](+), respectively. It is anticipated that the work in this area will not only help to facilitate improvements in mobility-based detection (IMS and MS), but also aid in the development and optimization of MS-based detection algorithms for TATP.

Characterizing the gas phase ion chemistry of an ion trap mobility spectrometry based explosive trace detector using a tandem mass spectrometer.

A commercial-off-the-shelf (COTS) ion trap mobility spectrometry (ITMS) based explosive trace detector (ETD) has been interfaced to a triple quadrupole mass spectrometer (MS/MS) for the purpose of characterizing the gas phase ion chemistry intrinsic to the ITMS instrument. The overall objective of the research is to develop a fundamental understanding of the gas phase ionization processes in the ITMS based ETD to facilitate the advancement of its operational effectiveness as well as guide the development of next generation ETDs. Product ion masses, daughter ion masses, and reduced mobility values measured by the ITMS/MS/MS configuration for a suite of nitro, nitrate, and peroxide containing explosives are reported. Molecular formulas, molecular structures, and ionization pathways for the various product ions are inferred using the mass and mobility data in conjunction with density functional theory. The predominant product ions are identified as follows: [TNT-H](-) for trinitrotoluene (TNT), [RDX+Cl](-) for cyclo-1,3,5-trimethylene-2,4,6-trinitramine (RDX), [NO(3)](-) for ethylene glycol dinitrate (EGDN), [NG+NO(3)](-) for nitroglycerine (NG), [PETN+NO(3)](-) for pentaerythritol tetranitrate (PETN), [HNO(3)+NO(3)](-) for ammonium nitrate (NH(4)NO(3)), [HMTD-NC(3)H(6)O(3)+H+Cl](-) for hexamethylene triperoxide diamine (HMTD), and [(CH(3))(2)CNH(2)](+) for triacetone triperoxide (TATP). The predominant ionization pathways for the formation of the various product ions are determined to include proton abstraction, ion-molecule attachment, autoionization, first-order and multi-order thermolysis, and nucleophilic substitution. The ion trapping scheme in the reaction region of the ITMS instrument is shown to increase predominant ion intensities relative to the secondary ion intensities when compared to non-ion trap operation.

Interfacing an ion mobility spectrometry based explosive trace detector to a triple quadrupole mass spectrometer.

Hardware from a commercial-off-the-shelf (COTS) ion mobility spectrometry (IMS) based explosive trace detector (ETD) has been interfaced to an AB/SCIEX API 2000 triple quadrupole mass spectrometer. To interface the COTS IMS based ETD to the API 2000, the faraday plate of the IMS instrument and the curtain plate of the mass spectrometer were removed from their respective systems and replaced by a custom faraday plate, which was fabricated with a hole for passing the ion beam to the mass spectrometer, and a custom interface flange, which was designed to attach the IMS instrument onto the mass spectrometer. Additionally, the mass spectrometer was modified to increase the electric field strength and decrease the pressure in the differentially pumped interface, causing a decrease in the effect of collisional focusing and permitting a mobility spectrum to be measured using the mass spectrometer. The utility of the COTS-ETD/API 2000 configuration for the characterization of the gas phase ion chemistry of COTS-ETD equipment was established by obtaining mass and tandem mass spectra in the continuous ion flow and selected mobility monitoring operating modes and by obtaining mass-selected ion mobility spectra for the explosive standard 2,4,6 trinitrotoluene (TNT). This analysis confirmed that the product ion for TNT is [TNT - H](-), the predominant collision-induced dissociation pathway for [TNT- H](-) is the loss of NO and NO(2), and the reduced mobility value for [TNT - H](-) is 1.54 cm(2)V(-1) s(-1). Moreover, this analysis was attained for sample amounts of 1 ng and with a resolving power of 37. The objective of the research is to advance the operational effectiveness of COTS IMS based ETD equipment by developing a platform that can facilitate the understanding of the ion chemistry intrinsic to the equipment.

C60 secondary ion mass spectrometry with a hybrid-quadrupole orthogonal time-of-flight mass spectrometer.

A hybrid quadrupole orthogonal time-of-flight mass spectrometer optimized for matrix-assisted laser desorption ionization (MALDI) and electrospray ionization has been equipped with a C 60 cluster ion source. This configuration is shown to exhibit a number of characteristics that improve the performance of traditional time-of-flight secondary ion mass spectrometry (TOF-SIMS) experiments for the analysis of complex organic materials and, potentially, for chemical imaging. Specifically, the primary ion beam is operated as a continuous rather than a pulsed beam, resulting in up to 4 orders of magnitude greater ion fluence on the target. The secondary ions are extracted at very low voltage into 8 mTorr of N 2 gas introduced for collisional focusing and cooling purposes. This extraction configuration is shown to yield secondary ions that rapidly lose memory of the mechanism of their birth, yielding tandem mass spectra that are identical for SIMS and MALDI. With implementation of ion trapping, the extraction efficiency is shown to be equivalent to that found in traditional TOF-SIMS machines. Examples are given, for a variety of substrates that illustrate mass resolution of 12,000-15,600 with a mass range for inorganic compounds to m/ z 40,000. Preliminary chemical mapping experiments show that with added sensitivity, imaging in the MS/MS mode of operation is straightforward. In general, the combination of MALDI and SIMS is shown to add capabilities to each technique, providing a robust platform for TOF-SIMS experiments that already exists in a large number of laboratories.

Energy deposition during molecular depth profiling experiments with cluster ion beams.

The role of the location of energy deposition during cluster ion bombardment on the quality of molecular depth profiling was examined by varying the incident angle geometry. Cholesterol films approximately 300 nm in thickness deposited onto silicon substrates were eroded using 40-keV C60(+) at incident angles ranging from 5 degrees to 73 degrees with respect to the surface normal. The erosion process was evaluated by determining at each incident angle the total sputtering yield of cholesterol molecules, the damage cross section of the cholesterol molecules, the altered layer thickness within the solid, the sputter yield decay in the quasi-steady-state sputter regime, and the interface width between the cholesterol film and the silicon substrate. The results show that the total sputtering yield is largest relative to the product of the damage cross section and the altered layer thickness at 73 degrees incidence, suggesting that the amount of chemical damage accumulated is least when glancing incident geometries are used. Moreover, the signal decay in the quasi-steady-state sputter regime is observed to be smallest at off-normal and glancing incident geometries. To elucidate the signal decay at near-normal incidence, an extension to an erosion model is introduced in which a fluence-dependent decay in sputter yield is incorporated for the quasi-steady-state regime. Last, interface width calculations indicate that at glancing incidence the damaged depth within the solid is smallest. Collectively, the measurements suggest that decreased chemical damage is not necessarily dependent upon an increased sputter yield or a decreased damage cross section but instead dependent upon depositing the incident energy nearer the solid surface resulting in a smaller altered layer thickness. Hence, glancing incident angles are best suited for maintaining chemical information during molecular depth profiling using 40-keV C60(+).

Relative Quantification of Cellular Sections with Molecular Depth Profiling ToF-SIMS Imaging.

We report the use of SIMS imaging to quantify the relative difference in the amount of lipid between two sections, the plasma membrane and the cytoplasm, of single cells from two different populations. Cells were each labeled with lipophillic dyes, frozen, fractured and analyzed in a ToF-SIMS mass spectrometer equipped with a 40 keV C(60) (+) ion source. In addition to identifying cells from separate populations, the lipophilic dyes can be used as a marker for the outer leaflet of the cell membrane and therefore as a depth finder. Here, we show that it is possible to compare the amount of lipids with particular headgroups in the cell membrane of a treated cell to the membrane of a control cell. Following erosion of the cell membranes, the amount of the two specific lipid head groups in the cytoplasm of the treated cell can be compared to those lipids in a control cell. Here we take the first step in this experimental design and display the ability to analyze multiple sections of frozen cells following a single fracture.

The effect of incident angle on the C(60) bombardment of molecular solids.

The effect of incident angle on the quality of SIMS molecular depth profiling using C(60) (+) was investigated. Cholesterol films of ~300 nm thickness on Si were employed as a model and were eroded using 40 keV C(60) (+) at an incident angle of 40° and 73° with respect to the surface normal. The erosion process was characterized by determining at each angle the relative amount of chemical damage, the total sputtering yield of cholesterol molecules, and the interface width between the film and the Si substrate. The results show that there is less molecule damage at an angle of incidence of 73° and that the total sputtering yield is largest at an angle of incidence of 40°. The measurements suggest reduced damage is not necessarily dependent upon enhanced yields and that depositing the incident energy nearer the surface by using glancing angles is most important. The interface width parameter supports this idea by indicating that at the 73° incident angle, C(60) (+) produces a smaller altered layer depth. Overall, the results show that 73° incidence is the better angle for molecular depth profiling using 40 keV C(60) (+).

Sputtering yields for C60 and Au3 bombardment of water ice as a function of incident kinetic energy.

The total sputtering yields for water ice due to kiloelectronvolt cluster bombardment have been measured and compared to the predictions made by the mesoscale energy deposition footprint (MEDF) model. For C60 bombardment, the experimental yield varies almost linearly from 820 water molecule equivalents at an incident kinetic energy of 10 keV to 10 100 water molecule equivalents at a kinetic energy of 120 keV. For Au3 bombardment, the experimental yield varies almost linearly from 630 water molecule equivalents at an incident energy of 10 keV and rises to 1200 water molecule equivalents at 25 keV. The MEDF model is used to calculate relative yield trends with respect to incident energy using short-time molecular dynamics simulations. The results of these calculations indicate that the model can effectively predict the yield trends observed for these two clusters in experiments, although there is a consistent overestimate of the predicted induced C60 yield. It is hypothesized that this overestimate can be explained by the absence of reactions and ionization processes in the current simulations. Despite this omission, experimental yield trends can be accurately predicted using relatively small amounts of computer time. The success of the model in predicting the yield of water from ice films using a variety of energies and projectiles suggests this approach may greatly aid in the optimization of experimental configurations.

Direct comparison of Au(3)(+) and C(60)(+) cluster projectiles in SIMS molecular depth profiling.

The sputtering properties of two representative cluster ion beams in secondary ion mass spectrometry (SIMS), C(60)(+) and Au(3)(+), have been directly compared. Organic thin films consisting of trehalose and dipalmitoylphosphatidylcholine (DPPC) are employed as prototypical targets. The strategy is to make direct comparison of the response of a molecular solid to each type of the bombarding cluster by overlapping the two ion beams onto the same area of the sample surface. The ion beams alternately erode the sample while keeping the same projectile for spectral acquisition. The results from these experiments are important to further optimize the use of cluster projectiles for SIMS molecular depth profiling experiments. For example, Au(3)(+) bombardment is found to induce more chemical damage as well as Au implantation when compared with C(60)(+). Moreover, C(60)(+) is found to be able to remove the damage and the implanted Au effectively. Discussions are also presented on strategies of enhancing sensitivity for imaging applications with cluster SIMS.

Surface sensitivity in cluster-ion-induced sputtering.

The ion beam-induced removal of thin water ice films condensed onto Ag and bombarded by energetic Au, Au2, Au3, and C60 projectiles is examined both experimentally and with molecular dynamics computer simulations. For water overlayers of thicknesses greater than 10 A, the yields of sputtered Ag+ secondary ions decay exponentially with increasing ice thickness, revealing characteristic decay lengths of 24, 20, 18, and 7.0 A, respectively. It is shown that these values manifest the characteristic depths of projectile energy loss, rather than escape depths of the sputtered Ag atoms through the water ice overlayer. Computer simulations show that the mechanism of ejection involves the sweeping away of overlayer water molecules, allowing for an unimpeded escape of ejected Ag atoms. The relevance of these data with respect to surface sensitivity in secondary ion mass spectrometry is discussed.

Secondary ion MS imaging of lipids in picoliter vials with a buckminsterfullerene ion source.

Investigation of the spatial distribution of lipids in cell membranes can lead to an improved understanding of the role of lipids in biological function and disease. Time-of-flight secondary ion mass spectrometry is capable of molecule-specific imaging of biological molecules across single cells and has demonstrated potential for examining the functional segregation of lipids in cell membranes. In this paper, standard SIMS spectra are analyzed for phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidylinositol, cholesterol, and sulfatide. Importantly, each of the lipids result in signature mass spectral peaks that allow them to be identified. These signature peaks are also useful for imaging experiments and are utilized here to simultaneously image lipids on a micrometer scale in picoliter vials. Because the low secondary ion signal achieved for lipids from an atomic primary ion source makes cell-imaging experiments challenging, improving signal with cluster primary ion sources is of interest. Here, we compare the secondary ion yield for seven lipids using atomic (Ga+ or In+) ion sources and a buckminsterfullerene (C60+) primary ion source. A 40-1000-fold improvement in signal is found with C60+ relative to the other two ion sources, indicating great promise for future cellular imaging applications using the C60+ probe.