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IUCrJ 5(1), 103 - 117 (2018). doi:10.1107/S2052252517017043, Previous proof-of-concept measurements on single-layer two-dimensional membrane-protein crystals performed at X-ray free-electron lasers (FELs) have demonstrated that the collection of meaningful diffraction patterns, which is not possible at synchrotrons because of radiation-damage issues, is feasible. Here, the results obtained from the analysis of a thousand single-shot, room-temperature X-ray FEL diffraction images from two-dimensional crystals of a bacteriorhodopsin mutant are reported in detail. The high redundancy in the measurements boosts the intensity signal-to-noise ratio, so that the values of the diffracted intensities can be reliably determined down to the detector-edge resolution of 4 Å. The results show that two-dimensional serial crystallography at X-ray FELs is a suitable method to study membrane proteins to near-atomic length scales at ambient temperature. The method presented here can be extended to pump–probe studies of optically triggered structural changes on submillisecond timescales in two-dimensional crystals, which allow functionally relevant large-scale motions that may be quenched in three-dimensional crystals., Published by IUCr, Chester

Facilitating the very short and intense pulses from an X-ray laser for the purpose of imaging small bioparticles carries the potential for structure determination at atomic resolution without the need for crystallization. In this study, experimental strategies for this idea are explored based on data collected at the Linac Coherent Light Source from 40 nm virus particles injected into a hard X-ray beam., This study explores the capabilities of the Coherent X-ray Imaging Instrument at the Linac Coherent Light Source to image small biological samples. The weak signal from small samples puts a significant demand on the experiment. Aerosolized Omono River virus particles of ∼40 nm in diameter were injected into the submicrometre X-ray focus at a reduced pressure. Diffraction patterns were recorded on two area detectors. The statistical nature of the measurements from many individual particles provided information about the intensity profile of the X-ray beam, phase variations in the wavefront and the size distribution of the injected particles. The results point to a wider than expected size distribution (from ∼35 to ∼300 nm in diameter). This is likely to be owing to nonvolatile contaminants from larger droplets during aerosolization and droplet evaporation. The results suggest that the concentration of nonvolatile contaminants and the ratio between the volumes of the initial droplet and the sample particles is critical in such studies. The maximum beam intensity in the focus was found to be 1.9 × 1012 photons per µm2 per pulse. The full-width of the focus at half-maximum was estimated to be 500 nm (assuming 20% beamline transmission), and this width is larger than expected. Under these conditions, the diffraction signal from a sample-sized particle remained above the average background to a resolution of 4.25 nm. The results suggest that reducing the size of the initial droplets during aerosolization is necessary to bring small particles into the scope of detailed structural studies with X-ray lasers.

X-ray free-electron lasers (XFELs) offer a new avenue to the structural probing of complex materials, including biomolecules. Delivery of precious sample to the XFEL beam is a key consideration, as the sample of interest must be serially replaced after each destructive pulse. The fixed-target approach to sample delivery involves depositing samples on a thin-film support and subsequent serial introduction via a translating stage. Some classes of biological materials, including two-dimensional protein crystals, must be introduced on fixed-target supports, as they require a flat surface to prevent sample wrinkling. A series of wafer and transmission electron microscopy (TEM)-style grid supports constructed of low-Z plastic have been custom-designed and produced. Aluminium TEM grid holders were engineered, capable of delivering up to 20 different conventional or plastic TEM grids using fixed-target stages available at the Linac Coherent Light Source (LCLS). As proof-of-principle, X-ray diffraction has been demonstrated from two-dimensional crystals of bacteriorhodopsin and three-dimensional crystals of anthrax toxin protective antigen mounted on these supports at the LCLS. The benefits and limitations of these low-Z fixed-target supports are discussed; it is the authors' belief that they represent a viable and efficient alternative to previously reported fixed-target supports for conducting diffraction studies with XFELs.

Bragg diffraction achieved from two-dimensional protein crystals using femtosecond X-ray laser snapshots is presented., X-ray diffraction patterns from two-dimensional (2-D) protein crystals obtained using femtosecond X-ray pulses from an X-ray free-electron laser (XFEL) are presented. To date, it has not been possible to acquire transmission X-ray diffraction patterns from individual 2-D protein crystals due to radiation damage. However, the intense and ultrafast pulses generated by an XFEL permit a new method of collecting diffraction data before the sample is destroyed. Utilizing a diffract-before-destroy approach at the Linac Coherent Light Source, Bragg diffraction was acquired to better than 8.5 Å resolution for two different 2-D protein crystal samples each less than 10 nm thick and maintained at room temperature. These proof-of-principle results show promise for structural analysis of both soluble and membrane proteins arranged as 2-D crystals without requiring cryogenic conditions or the formation of three-dimensional crystals.

FITC-albumin, Lsr-F, or fluorescent polystyrene latex particles were electrosprayed from aqueous buffer and subjected to dispersion by differential electrical mobility at atmospheric pressure. A resulting narrow size cut of singly charged molecular ions or particles was passed through a condensation growth tube collector to create a flow stream of small water droplets, each carrying a single ion or particle. The droplets were splash landed (impacted) onto a solid or liquid temperature controlled surface. Small pools of droplets containing size-selected particles, FITC-albumin, or Lsr-F were recovered, re-electrosprayed, and, when analyzed a second time by differential electrical mobility, showed increased homogeneity. Transmission electron microscopy (TEM) analysis of the size-selected Lsr-F sample corroborated the mobility observation.

Lasers have long played a critical role in the advancement of aerosol science. A new regime of ultrafast laser technology has recently be realized, the world's first soft x-ray free electron laser. The Free electron LASer in Hamburg, FLASH, user facility produces a steady source of 10 femtosecond pulses of 7–32 nm x-rays with 1012 photons per pulse. The high brightness, short wavelength, and high repetition rate (> 500 pulses per second) of this laser offers unique capabilities for aerosol characterization. Here we use FLASH to perform the highest resolution imaging of single PM2.5 aerosol particles in flight to date. We resolve to 35 nm the morphology of fibrous and aggregated spherical carbonaceous nanoparticles that existed for less than two milliseconds in vacuum. Our result opens the possibility for high spatial- and time-resolved single particle aerosol dynamics studies, filling a critical technological need in aerosol science.

To better understand the incorporation of membrane proteins into discoidal nanolipoprotein particles (NLPs) we have used atomic force microscopy (AFM) to image and analyze NLPs assembled in the presence of bacteriorhodopsin (bR), lipoprotein E4 n-terminal 22k fragment scaffold and DMPC lipid. The self-assembly process produced two distinct NLP populations: those containing inserted bR (bR-NLPs) and those that did not (empty-NLPs). The bR-NLPs were distinguishable from empty-NLPs by an average increase in height of 1.0 nm as measured by AFM. Streptavidin binding to biotinylated bR confirmed that the original 1.0 nm height increase corresponds to br-NLP incorporation. AFM and ion mobility spectrometry (IMS) measurements suggest that NLP size did not vary around a single mean but instead there were several subpopulations, which were separated by discrete diameters. Interestingly, when bR was present during assembly the diameter distribution was shifted to larger particles and the larger particles had a greater likelihood of containing bR than smaller particles, suggesting that membrane proteins alter the mechanism of NLP assembly.

We describe the first experimental measurements of aerodynamically focused particle beams using single particle image-charge detection. An aerodynamic lens produces particle beams, which at times is not aligned with the bore of the lens, thus complicating the process of aligning a particle beam with the focus of a laser beam. A key result of this work is the development of a non-optical technique for aiming a beam of particles in vacuum into the focus of a laser beam. In the present application, the laser beam is fixed in space by the geometry of a large stationary vacuum system and it is necessary to blindly aim a narrowly focused particle beam across the laser beam. Our aiming device is based on the non-destructive detection of electrically charged particles as they pass through a small metal tube that picks up the image charge of the transiting particle. Individual electrosprayed particles larger than 70 nm produce an electrical pulse that can be thresholded and counted. The duration of the detector signal provides a way to measure particle velocity and the amplitude of the signal is proportional to particle charge. The rate of particle injection into vacuum, single particle velocity and charge and particle beam shape and position can be measured with the charge detector. We show data for aiming and focusing electrosprayed polystyrene latex spheres ranging in size from 70 to 190 nm. Particle injection rates as high as 3000 per second and particle beam diameters as small as 250 μ m were achieved by using the charge detector to optimize the performance of the aerodynamic lens to focus the particles before they entered. We also describe how this detector and injector system will be implemented for real-time particle analysis for aerosol mass spectrometry and single particle X-ray diffractive imaging.

We are developing aerosol generating and processing methods for X-ray analyses of nanoscale materials using conventional synchrotron radiation sources and using the newly operational soft X-ray free-electron laser in Hamburg (FLASH) at the Deutsches Elektronen Synchrotron. Charge-reduced electrospray, differential mobility analysis and an electrostatic precipitator were used to prepare samples consisting of size-monodisperse spherical nanoparticles deposited on 20 nm thick silicon nitride foils supported by silicon frames. Ninety-seven and 102 nm diameter spheres were selected from a broader distribution of 98 nm spheres using differential mobility. We measured the size distribution of the spheres using forward scattering from 1.65 nm light at the Advanced Light Source (ALS) in Lawrence Berkeley National Laboratory and scanning electron microscopy (SEM). The full-width half maximum (FWHM) of the size distribution of the size-selected spheres was as narrow as 5.4 nm when measured by SEM, as compared to 16 nm for the non-size-selected distribution. Forward scattering measurements of the 97 nm diameter size-selected spheres fit a size distribution with a FWHM of 4 nm and allowed us to validate the methodology for use in future diffraction imaging experiments at FLASH.

Extremely intense and ultrafast X-ray pulses from free-electron lasers offer unique opportunities to study fundamental aspects of complex transient phenomena in materials. Ultrafast time-resolved methods usually require highly synchronized pulses to initiate a transition and then probe it after a precisely defined time delay. In the X-ray regime, these methods are challenging because they require complex optical systems and diagnostics. Here we propose and apply a simple holographic measurement scheme, inspired by Newton's 'dusty mirror' experiment, to monitor the X-ray-induced explosion of microscopic objects. The sample is placed near an X-ray mirror; after the pulse traverses the sample, triggering the reaction, it is reflected back onto the sample by the mirror to probe this reaction. The delay is encoded in the resulting diffraction pattern to an accuracy of one femtosecond, and the structural change is holographically recorded with high resolution. We apply the technique to monitor the dynamics of polystyrene spheres in intense free-electron-laser pulses, and observe an explosion occurring well after the initial pulse. Our results support the notion that X-ray flash imaging can be used to achieve high resolution, beyond radiation damage limits for biological samples. With upcoming ultrafast X-ray sources we will be able to explore the three-dimensional dynamics of materials at the timescale of atomic motion.

Protein oxidation is linked to cellular stress, aging, and disease. Protein oxidations that result in reactive species are of particular interest, since these reactive oxidation products may react with other proteins or biomolecules in an unmediated and irreversible fashion, providing a potential marker for a variety of disease mechanisms. We have developed a novel system to identify and quantitate, relative to other states, the sites of oxidation on a given protein. This presents a significant advancement over current methods, combining strengths of current methods and adding the abilities to multiplex, quantitate, and probe more modified amino acids. A specially designed Oxidation-dependent carbonyl-specific Element-Coded Affinity Mass Tag (O-ECAT), AOD, ((S)-2-(4-(2-aminooxy)-acetamido)-benzyl)-1,4,7,10-tetraazacyclododecane-N,N',N' ',N'''-tetraacetic acid, is used to covalently tag the residues of a protein oxidized to aldehyde or keto end products. O-ECAT can be loaded with a variety of metals, which yields the ability to generate mass pairs and multiplex multiple samples. The O-ECAT moiety also serves as a handle for identification, quantitation, and affinity purification. After proteolysis, the AOD-tagged peptides are affinity purified and analyzed by nanoLC-FTICR-MS (nanoliquid chromatography-Fourier transform ion cyclotron resonance-mass spectrometry), which provides high specificity in extracting coeluting AOD mass pairs with a unique mass difference and allows relative quantitation based on isotopic ratios. Using this methodology, we have quantified and mapped the surface oxidation sites on a model protein, recombinant human serum albumin (rHSA) in its native form (as purchased) and after FeEDTA oxidation both at the protein and amino acid levels. A variety of modified amino acid residues including lysine, arginine, proline, histidine, threonine, aspartic, and glutamic acids, were found to be oxidized to aldehyde and keto end products. The sensitivity of this methodology is shown by the number of peptides identified, twenty peptides on the native protein and twenty-nine after surface oxidation using FeEDTA and ascorbate. All identified peptides map to the surface of the HSA crystal structure, validating this method for identifying oxidized amino acids on protein surfaces. In relative quantitation experiments between FeEDTA oxidation and native protein oxidation, identified sites showed different relative propensities toward oxidation, independent of amino acid residue. This novel methodology not only has the ability to identify and quantitate oxidized proteins but also yields site-specific quantitation on a variety of individual amino acids. We expect to extend this methodology to study disease-related oxidation.

We present results from experiments at the Linac Coherent Light Source (LCLS) demonstrating that serial femtosecond crystallography (SFX) can be performed to high resolution (~2.5 Å) using protein microcrystals deposited on an ultra-thin silicon nitride membrane and embedded in a preservation medium at room temperature. Data can be acquired at a high acquisition rate using x-ray free electron laser sources to overcome radiation damage, while sample consumption is dramatically reduced compared to flowing jet methods. We achieved a peak data acquisition rate of 10 Hz with a hit rate of ~38%, indicating that a complete data set could be acquired in about one 12-hour LCLS shift using the setup described here, or in even less time using hardware optimized for fixed target SFX. This demonstration opens the door to ultra low sample consumption SFX using the technique of diffraction-before-destruction on proteins that exist in only small quantities and/or do not produce the copious quantities of microcrystals required for flowing jet methods.

Charge detection mass spectrometry (CDMS) has been used to rapidly analyze polymerase chain reaction (PCR) products with a minimum of sample cleanup after the PCR reaction. PCR products of 1525 base pairs (bp), 1982 bp and 2677 bp were detected by CDMS, where simultaneous measurement of the charge and velocity of an electrostatically accelerated ion allows a mass determination of the ion. We extended the investigation to a longer double-stranded, linear DNA sample by also analyzing linearized pBR322 (4361 bp). Positive ion mass spectra and ion charge-state distributions were obtained for these electrosprayed DNA samples. Instrumental conditions were studied using a commercially available, linearized pUC18 sample (2682 bp). Copyright © 1999 John Wiley & Sons, Ltd.

Energy-sensitive calorimetric detectors that operate at low temperatures (“cryogenic detectors”) have recently been applied for the first time as ion detectors in time-of-flight mass spectrometry. Compared to conventional, ionization-based detectors, which rely on secondary electron formation or the charge created in a semiconductor, cryogenic detectors measure low-energy solid state excitations created by a particle impact. This energy sensitivity of cryogenic detectors results in several potential advantages for TOF–MS. Cryogenic detectors are expected to have near 100% efficiency even for very large, slow-moving molecules, in contrast to microchannel plates whose efficiency drops considerably at large mass. Thus, cryogenic detectors could contribute to extending the mass range accessible by TOF–MS and help improving detection limits. In addition, the energy resolution provided by cryogenic detectors can be used for charge discrimination and studies of ion fragmentation, ion-detector interaction, and internal energies of large molecular ions. Cryogenic detectors could therefore prove to be a valuable diagnostic tool in TOF–MS. Here, we give a general introduction to the cryogenic detector types most applicable to TOF–MS including those types already used in several TOF–MS experiments. We review and compare the results of these experiments, discuss practical aspects of operating cryogenic detectors in TOF–MS systems, and describe potential near future improvements of cryogenic detectors for applications in mass spectrometry. © 1999 John Wiley & Sons, Inc., Mass Spec Rev 18: 155–186, 1999

The design and operation of a new type of ion trap provides a way to measure mass, charge, and velocity of large individual electrospray ions (>1 MDa, >250 charges) repeatedly during the time the ion is trapped. The trap consists of an image charge detection tube mounted between two ion mirrors. The mirrors are sets of parallel electrodes drilled with holes aligned with the bore of the detector tube. The device relies on voltages applied to the electrodes to establish symmetrically opposing ion focusing mirrors for the purpose of cycling ions through the charge detection tube many times. This design does not use magnetic or radio frequency fields to trap ions. Gating one of the mirrors to 0 V while maintaining voltages appropriate for reflecting ions on the opposite mirror allows an ion to pass through the holes in the mirror and enter the detector tube. A low-noise charge-sensitive amplifier, connected to the tube, reproduces the image charge of individual ions as they pass through the detector tube. Whe...

Characterizing intense, focused x-ray free electron laser (FEL) pulses is crucial for their use in diffractive imaging. We describe how the distribution of average phase tilts and intensities on hard x-ray pulses with peak intensities of 10(21) W/m(2) can be retrieved from an ensemble of diffraction patterns produced by 70 nm-radius polystyrene spheres, in a manner that mimics wavefront sensors. Besides showing that an adaptive geometric correction may be necessary for diffraction data from randomly injected sample sources, our paper demonstrates the possibility of collecting statistics on structured pulses using only the diffraction patterns they generate and highlights the imperative to study its impact on single-particle diffractive imaging.

Optics express 21(23), 28729(2013). doi:10.1364/OE.21.028729, Single shot diffraction imaging experiments via X-ray free- electron lasers can generate as many as hundreds of thousands of diffraction patterns of scattering objects. Recovering the real space contrast of a scat- tering object from these patterns currently requires a reconstruction process with user guidance in a number of steps, introducing severe bottlenecks in data processing. We present a series of measures that replace user guidance with algorithms that reconstruct contrasts in an unsupervised fashion. We demonstrate the feasibility of automating the reconstruction process by generating hundreds of contrasts obtained from soot particle diffraction experiments., Published by Soc., Washington, DC

Profiling structured beams produced by X-ray free-electron lasers (FELs) is crucial to both maximizing signal intensity for weakly scattering targets and interpreting their scattering patterns. Earlier ablative imprint studies describe how to infer the X-ray beam profile from the damage that an attenuated beam inflicts on a substrate. However, the beams in-situ profile is not directly accessible with imprint studies because the damage profile could be different from the actual beam profile. On the other hand, although a Shack-Hartmann sensor is capable of in-situ profiling, its lenses may be quickly damaged at the intense focus of hard X-ray FEL beams. We describe a new approach that probes the in-situ morphology of the intense FEL focus. By studying the translations in diffraction patterns from an ensemble of randomly injected sub-micron latex spheres, we were able to determine the non-Gaussian nature of the intense FEL beam at the Linac Coherent Light Source (SLAC National Laboratory) near the FEL focus. We discuss an experimental application of such a beam-profiling technique, and the limitations we need to overcome before it can be widely applied. © (2012) COPYRIGHT Society of Photo-Optical Instrumentation Engineers (SPIE). Downloading of the abstract is permitted for personal use only.

Results of coherent diffractive imaging experiments performed with soft X-rays (1-2 keV) at the Linac Coherent Light Source are presented. Both organic and inorganic nano-sized objects were injected into the XFEL beam as an aerosol focused with an aerodynamic lens. The high intensity and femtosecond duration of X-ray pulses produced by the Linac Coherent Light Source allow structural information to be recorded by X-ray diffraction before the particle is destroyed. Images were formed by using iterative methods to phase single shot diffraction patterns. Strategies for improving the reconstruction methods have been developed. This technique opens up exciting opportunities for biological imaging, allowing structure determination without freezing, staining or crystallization.

Lasers have long played a critical role in the advancement of aerosol science. A new regime of ultrafast laser technology has recently be realized, the world's first soft xray free electron laser. The Free electron LASer in Hamburg, FLASH, user facility produces a steady source of 10 femtosecond pulses of 7-32 nm x-rays with 10{sub 12} photons per pulse. The high brightness, short wavelength, and high repetition rate (>500 pulses per second) of this laser offers unique capabilities for aerosol characterization. Here we use FLASH to perform the highest resolution imaging of single PM2.5 aerosol particles in flight to date. We resolve to 35 nm the morphology of fibrous and aggregated spherical carbonaceous nanoparticles that existed for less than two milliseconds in vacuum. Our result opens the possibility for high spatialand time-resolved single particle aerosol dynamics studies, filling a critical technological need in aerosol science.

Background: Current methods for measuring the concentrations of lipoprotein particles and their distributions in particle subpopulations are not standardized. We describe here and validate a new gas-phase differential electrophoretic macromolecular mobility-based method (ion mobility, or IM) for direct quantification of lipoprotein particles, from small, dense HDL to large, buoyant, very-low-density lipoprotein (VLDL).Methods: After an ultracentrifugation step to remove albumin, we determined the size and concentrations of lipoprotein particles in serum samples using IM. Scan time is 2 min and covers a particle range of 17.2–540.0 Å. After scanning, data are pooled by totaling the particle number across a predetermined size range that corresponds to particular lipoprotein subclasses. IM results were correlated with those of standard methods for cholesterol and apolipoprotein analysis.Results: Intra- and interassay coefficients of variation for LDL particle size were Conclusions: The IM method provides accurate, reproducible, direct determination of size and concentration for a broad range of lipoprotein particles. Use of this methodology in studies of patients with cardiovascular disease and other pathologic states will permit testing of its clinical utility for risk assessment and management of these conditions.

Self-assembly of purified apolipoproteins and phospholipids results in the formation of nanometer-sized lipoprotein complexes, referred to as nanolipoprotein particles (NLPs). These bilayer constructs are fully soluble in aqueous environments and hold great promise as a model system to aid in solubilizing membrane proteins. Size variability in the self-assembly process has been recognized for some time, yet limited studies have been conducted to examine this phenomenon. Understanding the source of this heterogeneity may lead to methods to mitigate heterogeneity or to control NLP size, which may be important for tailoring NLPs for specific membrane proteins. Here, we have used atomic force microscopy, ion mobility spectrometry, and transmission electron microscopy to quantify NLP size distributions on the single-particle scale, specifically focusing on assemblies with 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and a recombinant apolipoprotein E variant containing the N-terminal 22 kDa fragment (E422k). Four discrete sizes of E422k/DMPC NLPs were identified by all three techniques, with diameters centered at approximately 14.5, 19, 23.5, and 28 nm. Computer simulations suggest that these sizes are related to the structure and number of E422k lipoproteins surrounding the NLPs and particles with an odd number of lipoproteins are consistent with the double-belt model, in which at least one lipoprotein adopts a hairpin structure.

We report the first successful reconstruction of the real space image from coherent X-ray diffraction patterns of membrane-supported nanoparticles using single ultrafast pulses. The particles consisted of 145-nm spherical polystyrene spheres that were size-selected by differential mobility analysis. We investigated the dependence of signal intensity on the number of spherical nanoparticles irradiated by single ultrafast pulses at the FLASH FEL facility. We demonstrate detection of as few as two 145-nm diameter particles irradiated by a single 32 nm fs-long FLASH pulse focused to 2.4 J cm −2 . In this case the noise in the diffraction pattern, due to photon-counting statistics and scattering from the supporting silicon nitride membrane, was the largest contributor to the recorded intensity. We were able to reconstruct high-resolution images of the nanoparticles using a strong scattering reference object to aid the phase retrieval of the coherent diffraction pattern. This method of reference-enhanced diffractive imaging may allow the imaging of weakly scattering objects at FLASH and other future X-ray FEL sources.

In nanotechnology, strategies for the creation and manipulation of nanoparticles in the gas phase are critically important for surface modification and substrate-free characterization. Recent coherent diffractive imaging with intense femtosecond X-ray pulses has verified the capability of single-shot imaging of nanoscale objects at suboptical resolutions beyond the radiation-induced damage threshold. By intercepting electrospray-generated particles with a single 15 femtosecond soft-X-ray pulse, we demonstrate diffractive imaging of a nanoscale specimen in free flight for the first time, an important step toward imaging uncrystallized biomolecules.

Theory predicts that with an ultrashort and extremely bright coherent X-ray pulse, a single diffraction pattern may be recorded from a large macromolecule, a virus, or a cell before the sample explodes and turns into a plasma. Here we report the first experimental demonstration of this principle using the FLASH soft X-ray free-electron laser. An intense 25 fs, 4 10^13 W/cm^2 pulse, containing 10^12 photons at 32 nm wavelength, produced a coherent diffraction pattern from a nano-structured non-periodic object, before destroying it at 60,000 K. A novel X-ray camera assured single photon detection sensitivity by filtering out parasitic scattering and plasma radiation. The reconstructed image, obtained directly from the coherent pattern by phase retrieval through oversampling, shows no measurable damage, and extends to diffraction-limited resolution. A three-dimensional data set may be assembled from such images when copies of a reproducible sample are exposed to the beam one by one., revtex, 6 pages, 4 figures

A simple mathematical technique for improving mass calibration accuracy of linear delayed extraction matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (DE MALDI-TOFMS) spectra is presented. The method involves fitting a parabola to a plot of Δm vs. mass data where Δm is the difference between the theoretical mass of calibrants and the mass obtained from a linear relationship between the square root of m/z and ion time of flight. The quadratic equation that describes the parabola is then used to correct the mass of unknowns by subtracting the deviation predicted by the quadratic equation from measured data. By subtracting the value of the parabola at each mass from the calibrated data, the accuracy of mass data points can be improved by factors of 10 or more. This method produces highly similar results whether or not initial ion velocity is accounted for in the calibration equation; consequently, there is no need to depend on that uncertain parameter when using the quadratic correction. This method can be used to correct the internally calibrated masses of protein digest peaks. The effect of nitrocellulose as a matrix additive is also briefly discussed, and it is shown that using nitrocellulose as an additive to the α-cyano-4-hydroxycinnamic acid (αCHCA) matrix does not significantly change initial ion velocity but does change the average position of ions relative to the sample electrode at the instant the extraction voltage is applied. Published in 2002 by John Wiley & Sons, Ltd.

The simultaneous measurement of the time-of-flight and the number of charges on each electrosprayed ion provides a new way to determine the mass of a whole intact virus. The weights of an icosahedral virus (rice yellow mottle virus) consisting of a single-stranded RNA surrounded by a homogeneous protein shell with a mass of 6.5×10

Changes in pollutant profiles observed in contaminated soils have been attributed to biological activity when in actuality abiotic processes caused pollutant removal.1 This sometimes casual implication of biodegradation is not acceptable, but understandable, because factors which cause the disappearance of pollutants are not always easy to identify. Several pollutant removal or transformation pathways are possible with most pollutants found in soil and these processes may or may not operate independently. Careful evaluation of bioremediation necessitates that all transformation and removal pathways are anticipated so that the pathways are either controlled or monitored but in practice this is generally not possible. The diminution in pollutant oxidation might track with several candidate loss mechanisms and when this occurs the real cause becomes problematic to pin point. Many factors disguise or prevent the effectual tracking of bioremediation processes among which are three general types of problems: (1) The estimate that only about 0.1% or less of the microorganisms living in soils are culturable leads to the conclusion that the possibility for unknown organisms participating in a biodegradation pathway is high. How should these imperceptible microorganisms be tracked? (2) The diversity of the microcosm is large, yet several different genera have been shown tomore » possess the same or very similar enzymes for degrading a pollutant. Cell counts of specific organisms, therefore may not track very well with the rate of loss or conversion of a pollutant. Should groups of organisms possessing similar biochemical reaction capabilities become the focus or is it more useful to track specific organisms? (3) Abiotic processes such as physical processes which transport pollutants away from a contamination site lead sometimes to the counterfactual conclusion that bioremediation occurred when in actuality the pollutant was diluted or dispersed heterogeneously. How do we handle a large number of samples so that significantly representative sampling and monitoring strategies are implementable? Diagnostic procedures based on identifying the activity of biodegrading organisms by using DNA-based procedures are attractive in light of these problems because they can be tuned to identify groups of organisms, specific organisms, and to detect signals that measure community activity.« less

We present results obtained with a novel mass spectrometer capable of determining the mass of multiply charged electrospray ions generated from samples of macromolecules in the megadalton (MDa) size range. The instrument utilizes a sensitive amplifier which can detect the charge on a single ion as it passes through a tube detector. A velocity measurement of an ion with known electrostatic energy provides the ion's mass-to-charge ratio. Simultaneous detection of the ion charge permits a mass assignment to be made for each ion. Electrospray ions of DNA and polymer molecules with masses greater than 1 x 10(6) Da and charge numbers (z) in excess of 425 e(-) are readily detected in this mass spectrometer. The weights of small particles were also measured. The on-axis single-ion detection configuration provides a duty cycle of nearly 100% and extends the practical application of electrospray mass spectrometry to the analysis of very large molecules with relatively inexpensive instrumentation.

these limitations for the analysis of whole viruses. Charge-detection mass spectrometry is a relatively new technique that circumvents limitations associated with detecting large, highly charged ions by making a simultaneous measurement of charge (z) and mass-to-charge (m/z) ratio for individual ions. The detection technique permits the mass analysis of electrospray ions with virtually unlimited mass [10‐14] and has been performed on both Fourier-transform ioncyclotron resonance [14] and time-of-flight [11‐13] mass analyzers. The electrospray ionization source used in these studies [11] produced an aerosol of highly-charged virus particles in the ion-evaporation region of the electrospray ion source at atmospheric pressure. Virus ions pass one at a time through a small metal flight tube attached to a charge-sensitive preamplifier that captures their image current, the magnitude of which is proportional to the ions charge. In addition, by measuring the time-of-flight [15] of each ion through the flight tube the m/z value can be determined. The mass of each ion is obtained from a combination of both the charge and m/z value, where the ion charge can typically be determined with a precision of better than 75 z (as demonstrated in previous experiments with microparticles). A more complete description of the experiments and the system is published else

Charge detection mass spectrometry (CD-MS) has been used to determine the mass of double-stranded, circular DNA and single-stranded, circular DNA in the range of 2500 to 8000 base pairs (1.5–5.0 MDa). Simultaneous measurement of the charge and velocity of an electrostatically accelerated ion allows a mass determination of the ion, with instrument calibration determined independently of samples. Positive ion mass spectra of electrosprayed commercial DNA samples supplied in tris(hydroxymethyl)ethylenediaminetetraacetic acid buffer, diluted in 50 vol. % acetonitrile, were obtained without cleanup of the sample. A CD mass spectrum constructed from 3000 ion measurements takes 10 min to acquire and yields the DNA molecular mass directly (mass resolution = 6). The data collected represent progress toward a more automatable alternative to sizing of DNA by gel electrophoresis. In addition to the mass spectra, CD-MS generates charge versus mass plots, which provide another means to investigate the creation and fate of large electrospray ions.

Characterizing supramolecular interactions offers significant challenges using NMR or crystallographic techniques either because of size limitations or the difficulty in forming suitable crystals, while mass spectrometry is largely limited to low resolution mass information. Here we report gas phase measurements of intact virus particles using electrospray ion mobility spectrometry with an accuracy in radial measurements that were sufficient to differentiate closely related species. In addition, measured diameters indicate that iscosahedral virus particles retain their structure in the gas phase as well as undergoing a slight compaction in the absence of solvent. Analysis of the human pathogen adenovirus represents the largest and most sophisticated biomolecular complex detected in the gas phase to date. These results, on a diverse set of viral systems, suggest that ion mobility spectrometry may have broad applications for the analysis of biological complexes.

The ultrafast, ultrabright X-ray pulses offered by a new generation of free-electron lasers is ushering in extraordinary new capabilities in X-ray science, with a wide range of applications in fundamental atomic-physics, ultrafast-chemistry and materials science.