Extending the laserspray ionization concept to produce highly charged ions at high vacuum on a time-of-flight mass analyzer

A new matrix compound, 2-nitrophloroglucinol, is reported which not only produces highly charged ions similar to electrospray ionization (ESI) under atmospheric pressure (AP) and intermediate pressure (IP) laserspray ionization (LSI) conditions but also the most highly charged ions so far observed for small proteins in mass spectrometry (MS) under high vacuum (HV) conditions. This new matrix extends the compounds that can successfully be employed as matrixes with LSI, as demonstrated on an LTQ Velos (Thermo) at AP, a matrix-assisted laser desorption/ionization (MALDI)-ion mobility spectrometry (IMS) time-of-flight (TOF) SYNAPT G2 (Waters) at IP, and MALDI-TOF Ultraflex, UltrafleXtreme, and Autoflex Speed (Bruker) mass spectrometers at HV. Measurements show that stable multiple charged molecular ions of proteins are formed under all pressure conditions indicating softer ionization than MALDI, which suffers a high degree of metastable fragmentation when multiply charged ions are produced. An important analytical advantage of this new LSI matrix are the potential for high sensitivity equivalent or better than AP-LSI and vacuum MALDI and the potential for enhanced mass selected fragmentation of the abundant highly charged protein ions. A second new LSI matrix, 4,6-dinitropyrogallol, produces abundant multiply charged ions at AP but not under HV conditions. The differences in these similar compounds ability to produce multiply charged ions under HV conditions is believed to be related to their relative ability to evaporate from charged matrix/analyte clusters.     

Multi-electron dynamics for neutralization of highly charged ions near surfaces

We present a simulation of the neutralization of highly charged ions in front of a lithium fluoride surface including the close-collision regime above the surface. The present approach employs a Monte-Carlo solution of the Liouville master equation for the joint probability density of the ionic motion and the electronic population of the projectile and the target surface. It includes single as well as double particle-hole (de)excitation processes and incorporates electron correlation effects through the conditional dynamics of population strings. For slow projectiles and normal incidence, the ionic motion depends sensitively on the interplay between image acceleration towards the surface and repulsion by an ensemble of positive hole charges in the surface (“trampoline effect”). For Ne¹⁰⁺ we find that image acceleration is dominant and no collective backscattering high above the surface takes place.     

Generation of highly charged peptide and protein ions by atmospheric pressure matrix-assisted infrared laser desorption/ionization ion trap mass spectrometry

We show that highly charged ions can be generated if a pulsed infrared laser and a glycerol matrix are employed for atmospheric pressure matrix-assisted laser desorption/ionization mass spectrometry with a quadrupole ion trap. Already for small peptides like bradykinin, doubly protonated ions form the most abundant analyte signal in the mass spectra. The center of the charge-state distribution increases with the size of the analyte. For example, insulin is detected with a most abundant ion signal corresponding to a charge state of four, whereas for cytochrome c, the 10 times protonated ion species produces the most intense signal. Myoglobin is observed with up to 13 charges. The high m/z ratios allow us to use the Paul trap for the detection of MALDI-generated protein ions that are, owing to their high molecular weight, not amenable in their singly protonated charge state. Formation of multiple charges critically depends on the addition of diluted acid to the analyte-matrix solution. Tandem mass spectra generated by collision-induced dissociation of doubly charged peptides are also presented. The findings allow speculations about the involvement of electrospray ionization processes in these MALDI experiments.     

On the formation of highly charged gaseous ions from unfolded proteins by electrospray ionization

Electrospray ionization (ESI) of native proteins results in a narrow distribution of low protonation states. ESI for these folded species proceeds via the charged residue mechanism. In contrast, ESI of unfolded proteins yields a wide distribution of much higher charge states. The current work develops a model that can account for this effect. Recent molecular dynamics simulations revealed that ESI for unfolded polypeptide chains involves protein ejection from nanodroplets, representing a type of ion evaporation mechanism (IEM). We point out the analogies between this IEM, and the dissociation of gaseous protein complexes after collisional activation. The latter process commences with unraveling of a single subunit, in concert with Coulombically driven proton transfer. The subunit then separates from the residual complex as a highly charged ion. We propose that similar charge equilibration events accompany the IEM of unfolded proteins, thereby causing the formation of high ESI charge states. A bead chain model is used for examining how charge is partitioned as protein and droplet separate. It is shown that protein ejection from differently sized ESI droplets generates a range of protonation states. The predicted behavior agrees well with experimental data.     

Detecting singly charged ions during field evaporation of tantalum at high temperatures

The field evaporation of tantalum from point emitters in a broad range of temperatures from T = 300 to 2500 K was studied using a static magnetic mass spectrometer equipped with a special field ion source. The room-temperature mass spectrum of field-evaporated particles displayed only the peaks of triply charged ions (Ta³⁺). As the temperature was increased, the charge of field-evaporated ions exhibited a decrease: at T ～ 1000 K, the peaks of doubly charged ions (Ta²⁺) prevailed. The peaks of singly charged ions (Ta⁺) were detected for the first time at temperatures in the interval 1900 K < T < 2500 K. The rate of evaporation of singly charged tantalum ions was several orders of magnitude lower than that of doubly charged ions.     

Producing highly charged ions without solvent using laserspray ionization: a total solvent-free analysis approach at atmospheric pressure

First examples of highly charged ions in mass spectrometry (MS) produced from the solid state without using solvent during either sample preparation or mass measurement are reported. Matrix material, matrix/analyte homogenization time and frequency, atmospheric pressure (AP) to vacuum inlet temperature, and mass analyzer ion trap conditions are factors that influence the abundance of the highly charged ions created by laserspray ionization (LSI). LSI, like matrix-assisted laser desorption/ionization (MALDI), uses laser ablation of a matrix/analyte mixture from a surface to produce ions. Preparing the matrix/analyte sample without the use of solvent provides the ability to perform total solvent-free analysis (TSA) consisting of solvent-free ionization and solvent-free gas-phase separation using ion mobility spectrometry (IMS) MS. Peptides and small proteins such as non-β-amyloid components of Alzheimer's disease and bovine insulin are examples in which LSI and TSA were combined to produce multiply charged ions, similar to electrospray ionization, but without the use of solvent. Advantages using solvent-free LSI and IMS-MS include simplicity, rapid data acquisition, reduction of sample complexity, and the potential for an enhanced effective dynamic range. This is achieved by more inclusive ionization and improved separation of mixture components as a result of multiple charging.     

Commercial intermediate pressure MALDI ion mobility spectrometry mass spectrometer capable of producing highly charged laserspray ionization ions

The first examples of highly charged ions observed under intermediate pressure (IP) vacuum conditions are reported using laser ablation of matrix/analyte mixtures. The method and results are similar to those obtained at atmospheric pressure (AP) using laserspray ionization (LSI) and/or matrix assisted inlet ionization (MAII). Electrospray ionization (ESI), LSI, and MAII are methods operating at AP and have been shown, with or without the use of a voltage or a laser, to produce highly charged ions with very similar ion abundance and charge states. A commercial matrix-assisted laser desorption/ionization ion mobility spectrometry (IMS) mass spectrometry (MS) instrument (SYNAPT G2) was used for the IP developments. The necessary conditions for producing highly charged ions of peptides and small proteins at IP appear to be a pressure drop region and the use of suitable matrixes and laser fluence. Ionization to produce these highly charged ions under the low pressure conditions of IP does not require specific heating or a special inlet ion transfer region. However, under the current setup, ubiquitin is the highest molecular weight protein observed. These findings are in accord with the need to provide thermal energy in the pressure drop region, similar to LSI and MAII, to improve sensitivity and extend the types of compounds that produce highly charged ions. The practical utility of IP-LSI in combination with IMS-MS is demonstrated for the analysis of model mixtures composed of a lipid, peptides, and a protein. Further, endogenous multiply charged peptides are observed directly from delipified mouse brain tissue with drift time distributions that are nearly identical in appearance to those obtained from a synthesized neuropeptide standard analyzed by either LSI- or ESI-IMS-MS at AP. Efficient solvent-free gas-phase separation enabled by the IMS dimension separates the multiply charged peptides from lipids that remained on the delipified tissue. Lipid and peptide families are exceptionally well separated because of the ability of IP-LSI to produce multiple charging.     

Real time analysis of brain tissue by direct combination of ultrasonic surgical aspiration and sonic spray mass spectrometry

Direct combination of cavitron ultrasonic surgical aspirator (CUSA) and sonic spray ionization mass spectrometry is presented. A commercially available ultrasonic surgical device was coupled to a Venturi easy ambient sonic-spray ionization (V-EASI) source by directly introducing liquified tissue debris into the Venturi air jet pump. The Venturi air jet pump was found to efficiently nebulize the suspended tissue material for gas phase ion production. The ionization mechanism involving solely pneumatic spraying was associated with that of sonic spray ionization. Positive and negative ionization spectra were obtained from brain and liver samples reflecting the primary application areas of the surgical device. Mass spectra were found to feature predominantly complex lipid-type constituents of tissues in both ion polarity modes. Multiply charged peptide anions were also detected. The influence of instrumental settings was characterized in detail. Venturi pump geometry and flow parameters were found to be critically important in ionization efficiency. Standard solutions of phospholipids and peptides were analyzed in order to test the dynamic range, sensitivity, and suppression effects. The spectra of the intact tissue specimens were found to be highly specific to the histological tissue type. The principal component analysis (PCA) and linear discriminant analysis (LDA) based data analysis method was developed for real-time tissue identification in a surgical environment. The method has been successfully tested on post-mortem and ex vivo human samples including astrocytomas, meningeomas, metastatic brain tumors, and healthy brain tissue.     

Adiabatic flow curves of metallic materials at high strain rates

Dynamic compression tests are carried out on Armco iron, Cr-V-steel, Ni-Cr-Mo-V-steel, an austenitic Ni-Cr-Mo-steel, tantalum, nickel and Ni₃Al and magnesium. The flow curves are analysed to determine the influence of the deformation energy which is tranformed into heat on the flow behaviour and mechancial stability. Not only the material properties but also the conditions of friction between the specimen and the compression tool are found to have a greate influence on the flow stress reduction and stability. High frictional forces promote mechanical instability of materials with low strain hardening and low strain rate sensitivity.     

Simultaneous analysis of 2-aminopyridine-derivatized neutral and sialylated oligosaccharides from human serum in the negative-ion mode by sonic spray ionization ion trap mass spectrometry

Neutral and acidic (sialylated) 2-aminopyridine-derivatized (PA) oligosaccharides were analyzed by using reversed-phase high-performance liquid chromatography/ion trap mass spectrometry (RP-HPLC/IT MS) with a sonic spray ionization (SSI) source. Under the RP-HPLC separation using a buffer of 1 mM ammonium acetate (pH4.3) at a flow rate of 0.2 mL/min, both PA-oligosaccharides in the negative-ion mode showed a comparable degree of ionization efficiency, differing from that of the positive-ion mode, which exhibits a wide gap between their ionization efficiencies. In addition, the ion intensities of both PA-oligosaccharides were higher in the negative-ion mode than in the positive-ion mode. These results strongly suggest that the negative-ion mode of SSI-MS is suitable for simultaneous analysis of neutral and acidic (sialylated) oligosaccharides in RP-HPLC/MS. In the present study, RP-HPLC/SSI-IT MS in the negative-ion mode was used in the analysis of PA-oligosaccharides from human serum and its usefulness was investigated. As a result, 32 neutral and sialylated PA-oligosaccharides from human serum were identified with differentiating isomeric oligosaccharides and relatively quantified by a single HPLC/MS run. This method is useful for simple and rapid analysis of the overall distribution of neutral and sialylated oligosaccharides in a complex sample such as serum.     

On the question of flow stress at high strain rates controlled by dislocation viscous flow

Moving dislocation experience a damping force because of their interactions with lattice phonons and electrons. Except at velocities close to the transverse sound velocity this force is proportional to the dislocation velocity. A high plastic strain rates dislocations may be required to move so quickly that dislocation damping forces determine the flow stress. In this note an estimate is made of strain rate at which dislocation damping becomes important. In the plastic flow stress range of 10 MPa to 100 MPa (for aluminum) the critical strain rate is of the order of 4 × 10⁵ s⁻¹to 10⁶ s⁻¹ if almost all the dislocation within a piece of metal are mobile during plastic straining. If only a small fraction of the dislocations during plastic straining is mobile, the critical strain rate is reduced by an amount equal to this fraction.     

Ultrarapid desalting of protein solutions for electrospray mass spectrometry in a microchannel laminar flow device

The adverse effects of nonvolatile salts on the electrospray (ESI) mass spectra of proteins and other biological analytes are a major obstacle for a wide range of applications. Numerous sample cleanup approaches have been devised to facilitate ESI-MS analyses. Recently developed microdialysis techniques can shorten desalting times down to several minutes, the bottleneck being diffusion of the contaminant through a semipermeable membrane. This work introduces an approach that allows the on-line desalting of macromolecule solutions within tens of milliseconds. The device does not employ a membrane; instead, it uses a two-layered laminar flow geometry that exploits the differential diffusion of macromolecular analytes and low molecular weight contaminants. To maximize desalting efficiency, diffusive exchange between the flow layers is permitted only for such a time as to allow full exchange of salt, while incurring minimal macromolecule exchange. Computer simulations and optical studies show that the device can reduce the salt concentration by roughly 1 order of magnitude, while retaining approximately 70% of the original protein concentration. Application of this approach to the on-line purification of salt-contaminated protein solutions in ESI-MS results in dramatic improvements of both the signal-to-noise ratio and the absolute signal intensity. However, efficient desalting requires the diffusion coefficients of salt and analyte to differ by roughly 1 order of magnitude or more. This technique has potential to facilitate high-throughput analyses of biological macromolecules directly from complex matrixes. In addition, it may become a valuable tool for process monitoring and for on-line kinetic studies on biological systems.     

Efficient electrophoretic method to remove neutral additives from protein solutions followed by mass spectrometry analysis

A mass spectrometry (MS)-compatible, isoelectric point-based separation method for removal of neutral additives from protein solutions is described. The separation is performed by electrophoretic migration and trapping using a device referred to as membrane separated wells for isoelectric focusing and trapping (MSWIFT). Electrophoretic separation in the MSWIFT device is fast; the entire process can be carried out in a matter of minutes, and it does not require further sample cleanup prior to MS analysis. Proof-of-concept experiments in which neutral additives (e.g., Triton X-100, Tween 20, poly(ethylene glycol)) are removed from protein solutions using the MSWIFT device followed by MS analysis are described. Coupling the MSWIFT separation with ion mobility MS provides additional separation via the gas phase and assists in achieving higher quality ESI mass spectra when small amounts of additives remain in solution.     

Metrological characteristics for thermoanemometer measuring transducers of fluid mass flow rates

Conclusions The variable-power mode can be obtained in two versions: by stabilizing the heating temperature or the difference between the heating temperature and that of the fluid.In the first version the temperature variation of the fluid affects the output signal both directly and indirectly: The direct influence is reduced as the heating temperature rises but the indirect influence is not dependent on it.In the second version (when stabilizing the temperature difference) the temperature variation of the fluid affects the output signal for the most part indirectly and the degree of this influence depends on the heating temperature, its character being different for various media.The transducer's accuracy can be substantially improved by correcting the output signal for the fluid's temperature variation. For complicated types of correction it is preferable to stabilize the heating temperature because then the number Pr_r is not affected by the form of the correcting function and it is described by a simpler expression.These conclusions are valid for any thermal flow-rate meters having a calibration characteristic that is expressed by Eq. (9).Translated from Izmeritel'naya Tekhnika, No. 12, pp. 47–48, December, 1980.     

A testing technique for concrete under confinement at high rates of strain

A testing device is presented for the experimental study of dynamic compaction of concrete under high strain rates. The specimen is confined in a metallic ring and loaded by means of a hard-steel Hopkinson pressure bar (80 mm diameter, 6 m long) allowing for the testing of specimens large enough regarding the aggregate size. The constitutive law for the metal of the ring being known, transverse gauges glued on its lateral surface allow for the measurement of the confining pressure. The hydrostatic and deviatoric responses of the specimen can then be computed. The proposed method is validated by several numerical simulations of tests involving a set of four different concrete-like behaviours and different friction coefficients between the cell and the specimen. Finally, three tests performed with the MB50 concrete at three different strain rates are processed with the method and are compared with literature results for the same material under quasi-static loadings.     

Tandem mass spectrometry of ribonuclease A and B: N-linked glycosylation site analysis of whole protein ions

Recently, an approach for the "top down" sequence analysis of whole protein ions has been developed, employing electrospray ionization, collision-induced dissociation, and ion/ion proton-transfer reactions in a quadrupole ion trap mass spectrometer. This approach has now been extended to an analysis of the [M + 12H]12+ to [M + 5H]5+ ions of ribonuclease A and its N-linked glycosylated analogue, ribonuclease B, to determine the influence of the posttranslational modification on protein fragmentation. In agreement with previous studies on the fragmentation of a range of protein ions, facile gas-phase fragmentation was observed to occur along the protein backbone at the C-terminal of aspartic acid residues, and at the N-terminal of proline, depending on the precursor ion charge state. Interestingly, no evidence was found for gas-phase deglycosylation of the N-linked sugar in ribonuclease B, presumably due to effective competition from the facile amide bond cleavage channels that "protect" the N-linked glycosidic bond from cleavage. Thus, localization of the posttranslational modification site may be determined by analysis of the "protein fragment ion mass fingerprint".     

Peculiarities of radiation from multiply charged ions near the Cherenkov threshold

The effect of a single electron capture (loss) by high-energy multiply charged ions on the characteristics of electromagnetic radiation emitted by ions near the Cherenkov threshold has been studied. The radiation is directed forward in a strongly smeared cone near the Cherenkov angle and exists in a subthreshold region of ion velocities.     

Laser desorption-atmospheric pressure chemical ionization mass spectrometry for the analysis of peptides from aqueous solutions

A recently reported ionization method, comprising an infrared (IR) laser pulse to desorb (LD) analyte species, followed by atmospheric pressure chemical ionization (APCI) with a corona discharge (LD-APCI) to effect ionization of the desorbed neutral analyte molecules, is described for the direct analysis of aqueous peptide solutions. The source employs a heated capillary atmospheric pressure (AP) inlet coupled to a quadrupole ion trap mass spectrometer and allows sampling under normal ambient air conditions. By use of the corona discharge, signals of the atmospheric pressure infrared matrix-assisted laser desorption/ionization (AP-IR-MALDI)-generated analyte protonated molecule were enhanced by factors as large as 1400. In addition, the acid modifier trifluoroacetic acid (TFA) was found to improve the AP-IR-MALDI-generated signal by a factor of approximately 10, whereas the LD-APCI generated signal yielded a 100-fold increase. In this study, the use of the corona discharge is described to enhance the analyte signal generated via AP-IR-MALDI and, as a tool, to probe the gas-phase neutral molecule population generated by the MALDI process. Finally, through the decoupling of desorption from ionization, implications regarding the application of LD-APCI for the direct analysis of numerous new analyte containing matrixes (e.g., polyacrylamide gel electrophoresis (PAGE), tissue, etc.) are discussed.     

Linear well-posedness of a hybrid inviscid/viscous flow problem with smooth and discontinuous solutions at the interface

Summary A hybrid inviscid/viscous flow problem is considered in this paper. The interface treatment is uniformly valid for problems with smooth and discontinuous solutions at the interface. It satisfies the requirement of conservation and nonlinear uniqueness for cases where the interface coincides with a shock. The well-posedness in the linear sense is studied for a two-dimensional hybrid inviscid/viscous flow problem.This is because the product of the small viscosity and a small gradient of the flow parameters outside the boundary layer is so small that the computer recognizes this product as zero.     

Structural characterization of protein tryptic peptides via liquid chromatography/mass spectrometry and collision-induced dissociation of their doubly charged molecular ions

The formation of multiply charged molecular ions via the field-assisted ion evaporation mechanism during electrospray ionization enables the use of an atmospheric pressure ionization quadrupole mass spectrometer system for characterizing biologically important peptides. The straightforward implementation of high-performance liquid chromatography (HPLC) into this new strategy to determine the molecular weight of tryptic peptides via the pneumatically assisted electrospray (ion spray) interface is presented. Examples utilizing both microbore (1.0 mm) and standard bore (4.6 mm) inside diameter columns are shown for the LC/MS molecular weight determination of tryptic peptides in methionyl-human growth hormone (met-hGH). Injected levels from 50 to 75 pmol of tryptic digest onto 1 mm i.d. HPLC columns provided full-scan LC/MS or LC/MS/MS results without postcolumn splitting of the effluent. When standard 4.6 mm i.d. HPLC columns were used, a 20:1 postcolumn split was utilized, which required from 1 to 5 nmol of injected tryptic digest for full-scan LC/MS or LC/MS/MS results. Collision-induced dissociation (CID) mass spectra resulting from either "infusion" or on-line LC/MS/MS analysis of the abundant doubly charged ions that predominate for tryptic peptides under electrospray conditions provided structurally useful sequence information for met-hGH and human hemoglobin tryptic digests. The slower mass spectrometer scan rate used during infusion of sample provides more accurate mass assignments than on-line LC/MS or LC/MS/MS, but the latter on-line experiments preclude ambiguities caused by matrix or component interferences. However, in some instances very weak CID product ions preclude complete tryptic peptide structural characterization based upon the CID data alone.(ABSTRACT TRUNCATED AT 250 WORDS)