Online measurement of phthalate-particulate matter interactions by membrane introduction mass spectrometry (MIMS).

To enable further study and assessment of indoor inhalation exposure risk, an online apparatus enabling measurement of semi-volatile compound partitioning on household particulates was developed. An example for use of the apparatus is described using dimethyl phthalate (DMP). The system employs direct measurement by membrane introduction mass spectrometry (MIMS). The MIMS system was calibrated using known gas phase DMP concentrations produced by gravimetrically calibrated permeation devices. The quantity of DMP sorbed by particles is described first using a model particle type, a reverse-phase liquid chromatography packing material, and then with a household dust sample. In addition, the desorption of semi-volatile compounds from a household dust sample was monitored using the apparatus, and characteristic fragment ion signals for phthalate compounds were observed.

Formation of a stable mimic of ambient particulate matter containing viable infectious respiratory syncytial virus and its dry-deposition directly onto cell cultures.

Epidemiological associations of worse respiratory outcomes from combined exposure to ambient particulate matter (PM) and respiratory viral infection suggest possible interactions between PM and viruses. To characterize outcomes of such exposures, we developed an in vitro mimic of the in vivo event of exposure to PM contaminated with respiratory syncytial virus (RSV). Concentration of infectious RSV stocks and a particle levitation apparatus were the foundations of the methodology developed to generate specific numbers of PM mimics (PM(Mimics)) of known composition for dry, direct deposition onto airway epithelial cell cultures. Three types of PM(Mimics) were generated for this study: (i) carbon alone (P(C)), (ii) carbon and infectious RSV (P(C+RSV)), and (iii) aerosols consisting of RSV (A(RSV)). P(C+RSV) were stable in solution and harbored infectious RSV for up to 6 months. Unlike A(RSV) infection, P(C+RSV) infection was found to be dynamin dependent and to cause lysosomal rupture. Cells dosed with PM(Mimics) comprised of RSV (A(RSV)), carbon (P(C)), or RSV and carbon (P(C+RSV)) responded differentially as exemplified by the secretion patterns of IL-6 and IL-8. Upon infection, and prior to lung cell death due to viral infection, regression analysis of these two mediators in response to incubation with A(RSV), P(C), or P(C+RSV) yielded higher concentrations upon infection with the latter and at earlier time points than the other PM(Mimics). In conclusion, this experimental platform provides an approach to study the combined effects of PM-viral interactions and airway epithelial exposures in the pathogenesis of respiratory diseases involving inhalation of environmental agents.

MALDI-MS monitoring of differential biomolecule secretion from human lung cells in vitro following incubation with <150 particles.

Knowledge of how the chemical composition of a given ambient particle affects varied biological response upon inhalation is of considerable interest regarding the pathogenesis of respiratory and cardiovascular diseases. Characterization of initiating events, using measurements of proinflammatory mediator differential expression by lung tissues, is an objective of our studies. Results demonstrating the capability to monitor changes in the secreted proteome of a human lung cell line culture dosed with <150 particles, for incubation periods ranging from 30 min to 24 h using matrix assisted laser desorption ionization (MALDI) mass spectrometry, are presented. Each population of particles was created within an electrodynamic balance to have the same size and chemical composition prior to being deposited onto a culture of A549 cells. The carbon particles, and the lipopolysaccharide (52 pg/particle) containing carbon particles, were 6.3 microm in diameter. Numerous biomolecules are observable in the mass spectra of supernatants of lung cells. The relative abundance of many of these biomolecules changes as a function of particle type and incubation time, suggesting the ion signal intensities observed are indicative of the relative differential expression of these compounds, some of which could be proinflammatory mediators.

Ion-induced nucleation in solution: promotion of solute nucleation in charged levitated droplets.

We have investigated the nucleation and growth of sodium chloride in both single quiescent charged droplets and charged droplet populations that were levitated in an electrodynamic levitation trap (EDLT). In both cases, the magnitude of a droplet's net excess charge (ions(DNEC)) influenced NaCl nucleation and growth, albeit in different capacities. We have termed the phenomenon ion-induced nucleation in solution. For single quiescent levitated droplets, an increase in ions(DNEC) resulted in a significant promotion of NaCl nucleation, as determined by the number of crystals observed. For levitated droplet populations, a change in NaCl crystal habit, from regular cubic shapes to dome-shaped dendrites, was observed once a surface charge density threshold of -9 x 10(-4) e.nm(-2) was surpassed. Although promotion of NaCl nucleation was observed for droplet population experiments, this can be attributed in part to the increased rate of solvent evaporation observed for levitated droplet populations having a high net charge. Promotion of nucleation was also observed for two organic acids, 2,4,6-trihydroxyacetophenone monohydrate (THAP) and alpha-cyano-4-hydroxycinnamic acid (CHCA). These results are of direct relevance to processes that occur in both soft-ionization techniques for mass spectrometry and to a variety of industrial processes. To this end, we have demonstrated the use of ion-induced nucleation in solution to form ammonium nitrate particles from levitated droplets to be used in in vitro toxicology studies of ambient particle types.

Dose-response studies involving controlled deposition of less than 100 particles generated and levitated in an ac trap onto lung cells, in vitro, and quantitation of ICAM-1 differential expression.

A developing area of interest regarding the relationship between the adverse health effects associated with particles suspended in the troposphere is an understanding of how particle chemical composition influences different biological outcomes. Described is the development and application of an apparatus and methodology wherein a known number of particles of tropospherically relevant chemical composition can be designed and levitated in an alternating current (ac) trap followed by their controlled deposition directly from the ac trap onto air-liquid interface cultured lung cells. A downstream biological response, differential upregulation of intercellular adhesion molecule (ICAM)-1, was measurable using fluorescence microscopy in the air-liquid interface human lung cell cultures even though the dose per culture was 0-100 lipopolysaccharide (LPS)-containing elemental carbon particles (52 pg LPS per 6.3 microm diameter particle). Fluorescence emission intensity data measured from a 1 mm2 area centered over the site of particle deposition were fitted using a least squares linear regression line. Because the total mass of each different compound comprising each of the particles delivered to the culture was known, the data generated with this methodology can be expressed as a pro-inflammation potential (in this case ICAM-1 expression) per particle number and composition. Also described is how this methodology affords opportunities to quantitatively study pro-inflammatory intercellular signaling leading to ICAM-1 expression at sites distal to the site of particle deposition.

Archiving and absolute quantitation of solutes separated by single charged droplet coulomb explosion.

The electrospray (ES) ion source relies on the transfer of low-volatility solutes to the gas phase as an outcome of coulomb explosion events of charged droplets generated by electrical atomization. Introduced here are two methods for archiving compounds separated by coulomb explosion of single droplets having net charge that had been levitated in an electrodynamic balance. We categorized compounds separated by the explosion as either material ejected, including progeny droplets, or the material retained in the main residue. The potential for this methodology is illustrated by (i) qualitative characterization of solute states, aqueous versus precipitated in the separated material, and (ii) absolute quantitation of solutes separated by such an event. For a droplet containing 5 x 10(7) 20-nm-diameter fluospheres, its first encountered coulomb explosion event resulted in the ejection of 1.70% of them. The capability to acquire such detailed information regarding the individual steps in the process of transferring low-volatility solutes to the gas phase in an ES ion source is essential to develop strategies for absolute quantitation in applications of ES mass spectrometry.

Apparatus for preparing mimics of suspended particles in the troposphere and their controlled deposition onto individual lung cells in culture with measurement of downstream biological response.

Inhalation exposure to particles <10 microm in size that are suspended in the troposphere (PM10) is a factor in respiratory and cardiovascular diseases. The extent of the injury, local to systemic inflammation, is dependent on the number, size, and composition of the particles to which an individual is exposed. The physical properties of and compounds on PM10 that are responsible for these adverse effects on human health are the subject of intense investigation. Here, we report a laboratory method that involved the creation of 1-120 particles per trial that were of known size and composition, followed by deposition of them directly onto individual human lung cells within a cell culture, and after an incubation period, a downstream biological response was measured. To illustrate this methodology, particles that each contained 50 pg of lipopolysaccharide were created and deposited onto individual cells over a region <0.36 mm2 within a genetically modified A549 cell culture. The biological readout was the relative expression of intercellular cell adhesion molecule 1 after 24 h of incubation using an immunocytochemistry assay. The apparatus and methodology introduced here enables studies at the interface between the relevant but diverse areas of atmospheric particle chemistry and lung cell biology to identify the chemical and physical factors of PM10 that cause/exacerbate respiratory and cardiovascular diseases by triggering various biological pathways.

Study of chemistry in droplets with net charge before and after Coulomb explosion: ion-induced nucleation in solution and implications for ion production in an electrospray.

Droplets with net charge are essential intermediaries in the production of gaseous ions in the electrospray (ES) ion source. There could be a wealth of knowledge regarding the chemistry that occurs in such droplets as a result of their violation of electroneutrality. Such information could lead to improved understanding of the ion generation process in an ES along with factors that affect it. The experiments performed involved the levitation of individually charged droplets that were, and were not, allowed to undergo Coulomb explosion while they remained levitated in an electrodynamic balance (EDB). Through examination of precipitates formed within the levitated droplets, it was observed that onset of NaCl precipitation was promoted in droplets (glycerol:water 9:1 v/v) that had mass-to-net-charge (m/z) ratio <-4.8 x 10(9) amu/e. This threshold m/z value is exceeded in essentially all droplets generated in an ES because it is above the calculated threshold for Coulomb explosion. This finding suggests that cluster formation in droplets having net charge could occur at solute concentrations lower than would be anticipated on the basis of homogeneous nucleation. The effect of large entities such as precipitates existing in the droplet on the dynamics of droplet Coulomb explosion was also examined. Using droplets whose initial size and magnitude of net charge were equivalent within experimental error but having different concentration of solutes, we showed that the m/z of their main residues following Coulomb explosion were different. Micrometer-size droplets containing 20 nm fluorescent beads that underwent Coulomb explosion resulted in main residues that had higher m/z for higher initial bead concentration in the starting solution (320 nM) when compared to main residues resultant from starting solutions having lower initial bead concentration (21 nM).

Interdomain and membrane interactions of CTP:phosphocholine cytidylyltransferase revealed via limited proteolysis and mass spectrometry.

CTP:phosphocholine cytidylyltransferase (CCT) is a multi-domain enzyme that regulates phosphatidylcholine synthesis. It converts to an active form upon binding cell membranes, and interdomain dissociations have been hypothesized to accompany this process. To identify these interdomain and membrane interactions, the tertiary structures of three forms of CCTalpha were probed by monitoring accessibility to proteases. Time-limited digestion with chymotrypsin or arginine C of soluble CCTalpha (CCT(sol)), phospholipid vesicle-bound CCT (CCT(mem)), and a soluble constitutively active CCT truncated at amino acid 236 generated complex mixtures of peptides that were resolved and identified by gel electrophoresis/immunoblotting and by matrix-assisted laser desorption/ionization-mass spectrometry, with or without coupling to capillary liquid chromatography. Identification of cleavage sites enabled assembly of peptide bond accessibility maps for each CCT form. Our results reveal a approximately 80-residue core within the catalytic domain (domain C) as the most inaccessible region in all three forms and the C-terminal phosphorylation domain as the most accessible. Membrane binding has little effect on the protease accessibility of these domains. To map the protease sites onto the catalytic domain, its three-dimensional structure was modeled from the atomic coordinates of glycerol-phosphate cytidylyltransferase (Protein Data Bank code 1COZ). The protease inaccessibility of most sites in domain C could be explained by burial or location within secondary structural elements. The accessibility of the N-terminal domain (domain N) was enhanced upon membrane binding. Residues Phe(234)-Leu(303) were inaccessible in CCT(mem), suggesting burial in the membrane. Surprisingly, residues Leu(274)-Leu(303) of this domain were also inaccessible in CCT(sol). We propose that this region is buried by interdomain contacts with domain N in CCT(sol). Membrane binding and burial of domain M in the lipid bilayer may disrupt this interaction, leading to increased exposure of sites in domain N.

Promotion of alpha-cyano-4-hydroxycinnamic acid and peptide cocrystallization within levitated droplets with net charge.

Many reactions occur as a result of charge imbalance within or between reactive species in reaction vessels that have zero net charge. Here, chemical processes taking place within reaction vessels having net excess charge were studied. For mass spectroscopists, a familiar example of vessels that defy electroneutrality are the charged droplets produced by an electrospray ion source. Evidence is presented that control of the magnitude of the net charge contained in a reaction vessel, in this case a levitated droplet, can be used to promote nucleation and crystal growth of a mixture of an organic acid, alpha-cyano-4-hydroxycinnamic acid (CHCA), with one or more peptides. This phenomenon was first observed during our ongoing development of wall-less sample preparation (WaSP), electrodynamic charged droplet processing methodology capable of creating micrometer-sized sample spots for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) from subnanoliter volumes of sample material. Peptide ion signal-to-noise (S/N) ratios obtained by MALDI-TOF-MS from sample spots created from droplets that had high relative magnitude of net charge were consistently greater than those detected from sample spots created from droplets that had lower net charge. To study this unexpected phenomenon further, WaSP methodology was developed to process different mass-to-charge (m/z) droplets levitated in an electrodynamic balance (EDB), facilitating their deposition onto different positions of a target to create arrays of droplet residues ordered from highest to lowest m/z. This capability allowed simultaneous levitation with subsequent separation of a population of droplets created from a single starting solution, but the droplets had varied magnitudes of net charge. After the droplets were ejected from the EDB and collected on a glass slide or MALDI plate, the solids contained in the deposited droplets were characterized using microscopy and MALDI-TOF-MS. Factors impacting the chemical processing in droplets having net excess charge levitated in an EDB are discussed with particular emphasis on their possible roles in the promotion of crystal nucleation and growth.

Preliminary investigation of electrodynamic charged droplet processing to couple capillary liquid chromatography with matrix-assisted laser desorption/ionization mass spectrometry.

Charged droplet processing methodology, that utilizes electrodynamic levitation technology to control the trajectories of picoliter volume charged droplets and deliver them to a target plate at atmospheric pressure, has been developed. Termed wall-less sample preparation (WaSP), this methodology offers several features that could prove beneficial to the preparation of sample spots from separation column effluents for matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) analysis. These features include solute pre-concentration factors of 10(1) to 10(3) due to volatile solvent evaporation prior to droplet deposition onto the target plate, high spatial accuracy of the deposition position of each processed droplet (+/-5 microm), and the ability to prepare sample spots as small as 20 microm in diameter from a single droplet. Here a new mode of operation of this methodology is described and used as an offline post-column pre-concentrating interface between capillary liquid chromatography (capLC) and a target plate for offline MALDI-MS. Using a fraction from the capLC separation of peptides produced by the proteolytic digestion of the protein cytidine 5'-triphosphate:phosphocholine cytidylyltransferase, MALDI sample spots were prepared using the dried-droplet method, direct piezoelectric droplet dispensing, and the processing of piezo-dispensed droplets by WaSP. The sample spot morphology was investigated using light microscopy, and peptide ion abundances produced by MALDI were measured using time-of-flight (TOF) MS. The advantages of developing an online capLC/WaSP interface with MALDI-MS in the future are discussed along with some of the challenges that may be encountered in such an endeavor.

Wall-less sample preparation of microm-sized sample spots for femtomole detection limits of proteins from liquid based UV-MALDI matrices.

Previously, we introduced wall-less sample preparation (WaSP), technology that involves the use of an electrodynamic balance (EDB) to prepare microm-sized sample spots for analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). In that work we demonstrated the detection of femtomole quantities of a low molecular weight peptide and a hydrophobic ester (both <600 Da). Here we use WaSP to test the hypothesis that the use of small sample spot sizes and an instrument equipped with delayed extraction would increase the analytical utility of liquid sample spots for peptide and protein (>2500 Da) analysis by UV-MALDI-TOF-MS (Sze et al.; J. Am. Soc. Mass Spectrom. 1998, 9, 166-174). To aid the optimization of preparing microm-sized sample spots by WaSP, optical microscopy and mass spectrometry were used to investigate nonvolatile solute concentration effects on droplet fission and sample spot size, modifications of the EDB electric field to control droplet ejection, and the use of multiple droplet deposition to increase sample loading. Also described is a rapid deposition mode of operation for WaSP that allows single droplets generated at 1 Hz to be levitated briefly ( approximately 500 ms) before being ejected autonomously and deposited as a concentrated sample spot with a spatial accuracy of +/-5 microm. To test the sensitivity of the method, one hundred glycerol droplets (270 pL each, 27 nL total) each containing 32 amol lysozyme were deposited on top of each other one-at-a-time to create a single sample spot. Using a mass spectrometer equipped with delayed extraction to analyze this sample spot, we verified the hypothesis of Sze et al. by achieving detection limits three orders of magnitude below that previously observed for the detection of a protein by UV-MALDI-TOF-MS with a chemical-doped liquid matrix sample preparation.

Plasticizer contamination from vacuum system O-rings in a quadrupole ion trap mass spectrometer.

The outgassing of plasticizers from Buna-N and Viton o-rings under vacuum lead to undesired ion-molecule chemistry in an Electrospray Quadrupole Ion Trap Mass Spectrometer. In experiments with the helium bath gas pressure >1.2 mTorr, or whenever analyte ions were stored for >100 ms, extensive loss of analyte ions by proton transfer or adduction with o-ring plasticizers bis(2-ethylhexyl) phthalate and bis(2-ethylhexyl) adipate occurred. A temporary solution to this contamination problem was found to be overnight refluxing in hexane of all the o-rings in the vacuum system. This procedure alleviated this plasticizer contamination for approximately 100 hours of operation. These results, and those that lead to identification of the contamination as plasticizers outgassing from o-rings are described.

Poly(ethylene glycol) doubly and singly cationized by different alkali metal ions: relative cation affinities and cation-dependent resolution in a quadrupole ion trap mass spectrometer.

Structural information of gas phase complexes of poly(ethylene glycol) (PEG) cationized by one or two different alkali metal ions is inferred from MS and MS/MS experiments performed with an electrospray quadrupole ion trap mass spectrometer. The rationale for selecting PEG was that its sites for cation binding are non-selective with respect to the repeating monomeric unit of the polymer, but there is selectivity with respect to the formation of an inner coordination sphere specific to each metal ion. The dissociation of [M1+ M2+ (EO23)], where EO23 = linear polymer of ethylene oxide, 23 units in length, resulted in loss of one of the alkali metal ions, with preference for loss of the larger cation, with no fragmentation of the PEG backbone for Na, K, Rb, and Cs. Li was not examined in this portion of the study. The selectivity for loss of the larger alkali metal ion was [Na+ K+ (EO23)] to [Na+ (EO23)] + K+ at 100%; [K+ Rb+ (EO23)] to [K+ (EO23)] + Rb+ at 93%; and [Rb+ Cs+ (EO23)] to [Rb+ (Eo23)] + Cs+ at 99%. The resolution of [M+ (EOx)] for x = 20-30 was dependent on the alkali metal ion, with the highest resolution observed for Cs+ and the lowest for Na+. These results are discussed with respect to the packing of the oxygen atoms on PEG (M.W.(avg) = 1000) around an alkali metal ion of different radius, and how this packing leads to an ensemble of unique structures, and therefore mobilities for [M+ (EOx)].

MALDI-TOF-MS analysis of droplets prepared in an electrodynamic balance: "wall-less" sample preparation.

Methodology enabling mass spectral analysis of the composition of droplet(s) prepared in an electrodynamic balance (EDB) by matrix-assisted laser desorption/ionization (MALDI) is described. The dc field surrounding the electrodynamic balance was manipulated to eject single droplets at a time from the EDB thereby causing their deposition onto a MALDI sample plate precoated with matrix. When the laser was directed onto the droplet(s) and held stationary, marked gains in the signal-to-noise and signal-to-background ratios were realized with each subsequent mass spectrum due to the suppression of matrix cluster ion formation. Optical microscopy of the plate, after 1024 laser shots were fired at eight droplets that had been deposited one on top of the other, revealed a residual island of droplet matter (area approximately 3.1 x 10(-9) m2) inside the region where the crystalline matrix had been ablated away within the laser spot (area approximately 1.6 x 10(-8) m2). Removing the predried crystalline matrix layer and, instead, adding matrix into the starting solution was found to be a more effective means of suppressing matrix cluster ion formation. The chemical composition of the droplet(s) prepared in the EDB is discussed with regard to sample preconcentration, the images of the laser spot after MALDI, matrix cluster ion suppression, and the possibility for improved quantitation and detection limits by MALDI-TOF-MS.