Pulsed hydrogen/deuterium exchange MS/MS for studying the relationship between noncovalent protein complexes in solution and in the gas phase after electrospray ionization

Electrospray ionization mass spectrometry (ESI-MS) has become a standard method for monitoring noncovalent protein-protein interactions. Studies employing this approach tend to operate on the premise that the ionic species observed in the mass spectrum directly reflect the corresponding solution-phase protein quaternary structures. However, dissociation or clustering events taking place during ESI may lead to disparities between the ions observed in the mass spectrum and the protein binding state in bulk solution. Recognizing the occurrence of dissociation or clustering artifacts is not straightforward, leading to possible ambiguities in the interpretation of ESI-MS data. This work employs on-line pulsed hydrogen-deuterium exchange (HDX) for probing the origin of various species in the ESI mass spectrum of hemoglobin. In addition to the canonical hemoglobin tetramer, ESI-MS reveals the presence of monomers, dimers, hexamers, and octamers. Tandem mass spectrometry (MS/MS) is used for extracting HDX levels in a subunit-specific manner. Dimeric species exhibit exchange levels that are significantly above those of the tetramer. Monomeric hemoglobin subunits are labeled to an even greater extent. This HDX pattern implies that monomers and dimers do not represent dissociation artifacts generated during ESI. Instead, they are derived from preexisting solution-phase structures. In contrast, hexamers and octamers exhibit HDX levels that resemble those of the tetramer, thus identifying these larger species as nonspecific clustering artifacts. Overall, it appears that the pulsed HDX MS/MS approach introduced in this work represents a widely applicable tool for deciphering the relationship between ESI mass spectra and protein quaternary structures in solution.     

Atmospheric pressure laser ionization. An analytical technique for highly selective detection of ultralow concentrations in the gas phase

In this contribution a new analytical technique is presented for the direct mass spectrometric (MS) detection of gas-phase trace species at atmospheric pressure. Employing resonance-enhanced multiphoton ionization (REMPI) close to the inlet nozzle orifice, i.e., at high molecule densities, the sensitivity of the instrument has been increased by up to 3 orders of magnitude as compared to conventional REMPI-MS with ionization in a differentially coupled ion source. Furthermore, adiabatic cooling, resonant ionization, and mass-selective detection establish a highly selective analytical technique. Several atmospherically relevant compounds were investigated. The current detection limit for NO is 0.9 pptv, for acetaldehyde 1.7 pptv, for CO 15 pptv, and for 2,5-dichlorotoluene 12 pptv. We discuss APLI with regard to applications in medical and environmental research.     

Comparison of atmospheric pressure chemical ionization, electrospray ionization, and atmospheric pressure photoionization for the determination of cyclosporin A in rat plasma

Atmospheric pressure chemical ionization was compared with electrospray ionization and atmospheric pressure photoionization (APPI) as an interface of high-performance liquid chromatography (HPLC)-tandem mass spectrometry (MS/MS) for the determination of cyclosporin A (CsA) in biological fluids in support of in vivo pharmacodynamic studies. These ion sources were investigated in terms of their suitability and sensitivity for the detection of CsA. The effects of the eluent flow rate and composition as well as the nebulizer temperatures on the photoionization efficiency of CsA in the positive ion mode under normal-phase HPLC conditions were explored. The ionization mechanism in the APPI environment with and without the use of the dopant was studied using two test compounds and a few solvent systems employed for normal-phase chromatography. The test compounds were observed to be ionized mainly by proton transfer with the self-protonated solvent molecules produced through photon irradiation. Furthermore, ion suppression due to sample matrix interference in the normal-phase HPLC-APPI-MS/MS system was monitored by the postcolumn infusion technique. The applicability of these proposed HPLC-API-MS/MS approaches for the determination of CsA at low nanogram per milliliter levels in rat plasma was examined. These proposed methods were then compared with respect to specificity, linearity, detection limit, and accuracy.     

Atmospheric pressure chemical ionization source. 1. Ionization of compounds in the gas phase

A novel chemical ionization source for organic mass spectrometry is introduced. This new source uses a glow discharge in the flowing afterglow mode for the generation of excited species and ions. The direct-current gas discharge is operated in helium at atmospheric pressure; typical operating voltages and currents are around 500 V and 25 mA, respectively. The species generated by this atmospheric pressure glow discharge are mixed with ambient air to generate reagent ions (mostly ionized water clusters and NO+), which are then used for the ionization of gaseous organic compounds. A wide variety of substances, both polar and nonpolar, can be ionized. The resulting mass spectra generally show the parent molecular ion (M+ or MH+) with little or no fragmentation. Proton transfer from ionized water clusters has been identified as the main ionization pathway. However, the presence of radical molecular ions (M+) for some compounds indicates that other ionization mechanisms are also involved. The analytical capabilities of this source were evaluated with a time-of-flight mass spectrometer, and preliminary characterization shows very good stability, linearity, and sensitivity. Limits of detection in the single to tens of femtomole range are reported for selected compounds.     

Comparison of electrospray,atmospheric pressure chemical ionization,and atmospheric pressure photoionization in the identification of apomorphine,dobutamine, and entacapone phase II metabolites in biological samples

The applicability of different ionization techniques, electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), and a novel atmospheric pressure photoionization (APPI), were tested for the identification of the phase II metabolites of apomorphine, dobutamine, and entacapone in rat urine and in vitro incubation mixtures (rat hepatocytes and human liver microsomes). ESI proved to be the most suitable ionization method; it enabled detection of 22 conjugates, whereas APCI and APPI showed only 12 and 14 conjugates, respectively. Methyl conjugates were detected with all ionization methods. Glucuronide conjugates were ionized most efficiently with ESI. Only some of the glucuronides detected with ESI were detected with APCI and APPI. Sulfate conjugates were detected only with ESI. MS/MS experiments showed that the site of glucuronidation or sulfation could not be determined, since the primary cleavage was a loss of the conjugate group (glucuronic acid or SO3), and no site-characteristic product ions were formed. However, it may be possible to determine the site of methylation, since methylated products are more stable than glucuronides or sulfates. Furthermore, the loss of CH3 is not necessarily the primary cleavage, and site characteristic products may be formed. Identification and comparison of conjugates formed from the current model drugs were successfully analyzed in different biological specimens of common interest to biomedical research. A fairly good relation was obtained between the data from in vivo and in vitro models of drug metabolism.     

Direct quantitative analysis of organic compounds in the gas and particle phase using a modified atmospheric pressure chemical ionization source in combination with ion trap mass spectrometry

A slightly modified atmospheric pressure chemical ionization source is employed for direct quantitative analysis of volatile or semivolatile organic compounds in air. The method described here is based on the direct introduction of an analyte in the gas or particle phase, or both, into the ion source of a commercial ion trap mass spectrometer. For quantitation, a standard solution is directly transferred into the vaporizer unit of the ion source via a deactivated fused-silica capillary by using the sheath liquid syringe pump, which is part of the mass spectrometer. The standard addition procedure is conducted by varying the pump rate of a diluted solution of the standard compound in methanol/water. A N2 sheath gas flow is applied for optimal vaporization and mixing with the analyte gas stream. By performing detailed reagent ion monitoring experiments, it is shown that the relative signal intensity of [M + H]+ ions is dependent on the relative humidity of the analyte gas stream as well as the composition and concentration of CI reagent ions. The method is validated by a comparison of the standard addition results with a calibration test gas of known concentration. To demonstrate the potential of atmospheric pressure chemical ionization mass spectrometry as a quantitative analytical technique for on-line investigations, a tropospherically relevant reaction is carried out in a 493-L reaction chamber at atmospheric pressure and 296 K in synthetic air at 50% relative humidity. Finally, the applicability of the technique to rapidly differentiate between analytes in the gas and particle phase is demonstrated.     

Thermodynamic investigations of the gas phase reaction of SiH4 and O2 at atmospheric pressure

In this paper we present the results of a thermodynamic study of the gas phase reaction between SiH₄ and O₂ in terms of the gas phase composition and reaction efficiency.

A comparison of the theoretical findings with the experimental results suggests that above 400 °C the reaction tends to be thermodynamically controlled with increasing temperature, while below 400 °C a large deviation from the thermodynamic prediction exists.     

A REMPI method for the ultrasensitive detection of NO and NO2 using atmospheric pressure laser ionization mass spectrometry

We report on the development of a quasi-simultaneous highly selective method for NO and NO2 detection at the ultratrace level. Atmospheric pressure laser ionization (APLI), recently introduced by our group, is used to detect both compounds at low parts per trillion by volume (pptv) mixing ratios. APLI is based on resonance-enhanced multiphoton ionization mass spectrometry. Two-color pump-probe experiments employing a single excimer pumped dye laser combination allow for the ultrasensitive measurement of NO and NO2 within a narrow range of maximum pumping efficiency of the laser dye Coumarin 120. NO is detected via excitation of the long-lived A 2sigma+ (nu' = 1) level at 215.36 nm and subsequently ionized with 308-nm radiation provided by the excimer pump laser. NO2 is ionized after double resonant excitation of the A2B1 and 3psigma manifolds in a (1 + 1' + 1(')) process using 431.65 + 308 nm. The selectivity of the NO measurement exceeds 2,000 with respect to NO2 and N2O5. For NO2, a selectivity of >3,000 with respect to N2O5 and organic nitrates is observed. The current APLI detection limit of NO and NO2 is 0.5 and 5 pptv, respectively, with a 20-s integration time.     

Host-guest chemistry in the gas phase: selected fragmentations of CB[6]-peptide complexes at lysine residues and its utility to probe the structures of small proteins

The gas phase host-guest chemistry between cucurbit[6]uril (CB[6]) and peptide is investigated using electrospray ionization mass spectrometry (ESI-MS). CB[6] exhibits a high preference to interacting with a Lys residue in a peptide forming a CB[6]-peptide complex. Collisionally activated CB[6] complexes of peptides yield a common highly selective fragment product at m/z 549.2, corresponding to the doubly charged CB[6] complex of 5-iminiopentylammonium (5IPA). The process involves the formation of an internal iminium ion, which results from further fragments to an a-type ion from a y-type ion, and the resulting 5IPA ion threads through CB[6]. Numerous peptides are investigated to test the generality of the observed unique host-guest chemistry of CB[6]. Its potential utility in probing protein structures is demonstrated using CB[6] complexes of ubiquitin. Low-energy collision induced dissociation yields CB[6] complex fragments, and further MS(n) spectra reveal details of the CB[6] binding sites, which allow us to deduce the protein structure in the solution phase. The mechanisms and energetics of the observed reactions are evaluated using density functional theory calculations.     

Rem and reels characterization of MgO (100) surfaces. A Technique for studying the cleaving mechanisms and reactions of crystal surfaces

Reflection electron microscopy (REM) is used as a technique for imaging MgO surfaces cleaved by different methods. Several distinct surface structures of MgO (100) have been observed, and the possibility of relating the surface structure images with the breaking mechanisms of the surfaces has been shown. The surface atomic inner-shell excitations have been observed with reflection electron energy-loss spectroscopy (REELS), and the structure modulation of the near-edge fine structure of the O K and Mg K edges is considered to be due to changes in the surface density of states. The REM imaging is capable of providing the nucleation processes of the reaction products on the surfaces in the solid-liquid reactions and the associated relations with the surface structures.     

A method for determining the states of gas molecules and radicals participating in heterogeneous chemical reactions

A new method is proposed for monitoring the participation of free, preadsorbed, and chemisorbed gas species in chemical reactions at a solid-gas interface. The phenomena of energy exchange in the layer of adsorbed radicals, leading to an increase in the rate of heterogeneous recombination processes, were observed.     

A method for energy estimation and mass composition determination of primary cosmic rays at the Chacaltaya observation level based on the atmospheric Cherenkov light technique

A new method for energy and mass composition estimation of primary cosmic rays based on the atmospheric Cherenkov light flux in extensive air showers (EAS) is proposed. The Cherenkov light flux in EAS initiated by primary protons and iron nuclei is simulated with the CORSIKA 5.62 code for the Chacaltaya observation level (536 g/cm²) in the energy range 10 TeV–10 PeV. An adequate model for the lateral distribution of Cherenkov light in showers is obtained. Using the model and a solution for the overdetermined system of nonlinear equations based on the Gauss–Newton method with autoregularization, two different detector arrangements are compared. The accuracies in energy and shower axis determination are studied and the corresponding selection criteria are proposed. An approximation with a nonlinear fit is obtained and the energy dependence of the proposed model parameters is studied. A detailed study of the model parameters as a function of the primary energy is made. This permits, taking into account the properties of the proposed method and the strong nonlinearity of the model, a distinction to be made between proton and iron primaries. The detector response for the detector sets is simulated and the accuracies in energy determination are calculated. In addition, the accuracies in shower axis determination are studied and criteria for shower axis position estimation are proposed.     

A simple and robust method for determining the number of basic sites in peptides and proteins using electrospray ionization mass spectrometry

A solution additive has been discovered that can be used to measure the number of basic sites in a peptide or protein using electrospray ionization (ESI) mass spectrometry. Addition of millimolar amounts of perchloric acid (HClO(4)) to aqueous solutions that contain peptides or proteins results in the noncovalent adduction of HClO(4) molecules to the multiply charged ions formed by ESI. For 18 oligopeptides and proteins, ranging in molecular weight from 0.5 to 18.3 kDa, the sum of the number of protons plus maximum number of HClO(4) molecules adducted to the lower charge state ions is equal to the number of basic sites in the molecule. This method provides a rapid means of obtaining information about the composition of a peptide or protein and does not require high-resolution measurements or any instrumental or experimental modifications.     

鞘氨醇单胞菌降解多环芳烃菲过程中菌体全蛋白提取方法的优化及其蛋白的差异表达

采用平板升华技术从厦门博坦油码头表层海水样品中筛选获得一株高效降解多环芳烃(PAHs)的菌,经16S rDNA分子鉴定,该菌株归属鞘氨醇单胞菌属,定名为Sphin-gomonassp.strain H。Strain H在24h内对初始浓度为50mg/L的多环芳烃菲的降解率可达94%。以strain H为模式菌株,对其在降解过程中菌体全蛋白的提取进行了优化,以获得高质量的降解全程各个阶段具有代表性的菌体蛋白样品。结果表明,超声-三氯乙酸(TCA)/丙酮沉淀的方法可有效去除中间代谢产物的干扰,获得纯度较高的蛋白样品。对以此方法提取的蛋白样品,通过双向电泳技术比较了strain H在有和没有菲诱导条件下的蛋白质表达差异,获得17个差异蛋白点。这些差异蛋白点的获得将为进一步分析参与PAHs降解的关键酶系和探索菲的代谢途径奠定实验基础。     

Estimation of air/coating distribution coefficients for solid phase microextraction using retention indexes from linear temperature-programmed capillary gas chromatography. Application to the sampling and analysis of total petroleum hydrocarbons in air

The paper describes a method to quantify hydrocarbons in air exclusively on the basis of chromatographic parameters without the need for calibration. A simple technique is presented to estimate distribution coefficients (K) between air and the poly(dimethylsiloxane) solid phase microextraction (SPME) fiber coating using the linear temperature-programmed retention index system (LTPRI). There is a linear relationship (r(2) = 0.99989) between the log K for a series of n-alkanes and LTPRI, thus providing a means by which establishing a K for any peak in a chromatogram is possible given its published or experimentally determined LTPRI. This alternative approach to establishing K values significantly enhances and simplifies the use of SPME for sampling and analyzing air for quantification of compounds without the need for fiber calibration. Analysis of a group of 29 isoparaffinic compounds and a group of 33 aromatic compounds showed excellent agreement between their theoretical air to fiber distribution coefficients based on LTPRI and the experimentally obtained distribution coefficients. In addition, for a very complex mixture of organics such as gasoline, SPME can establish a total petroleum hydrocarbon in air level using LTPRI. This method was carefully evaluated, and the results were essentially identical between standard procedures and the proposed simple procedure described in the paper.