Sample Deposition Method Research Articles

Versatile microporous polymer-based supports for serial macromolecular crystallography.

Serial data collection has emerged as a major tool for data collection at state-of-the-art light sources, such as microfocus beamlines at synchrotrons and X-ray free-electron lasers. Challenging targets, characterized by small crystal sizes, weak diffraction and stringent dose limits, benefit most from these methods. Here, the use of a thin support made of a polymer-based membrane for performing serial data collection or screening experiments is demonstrated. It is shown that these supports are suitable for a wide range of protein crystals suspended in liquids. The supports have also proved to be applicable to challenging cases such as membrane proteins growing in the sponge phase. The sample-deposition method is simple and robust, as well as flexible and adaptable to a variety of cases. It results in an optimally thin specimen providing low background while maintaining minute amounts of mother liquor around the crystals. The 2 × 2 mm area enables the deposition of up to several microlitres of liquid. Imaging and visualization of the crystals are straightforward on the highly transparent membrane. Thanks to their affordable fabrication, these supports have the potential to become an attractive option for serial experiments at synchrotrons and free-electron lasers.

High-Mass Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry for Absolute Quantitation of Noncovalent Protein-Protein Binding Interactions.

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is a robust and powerful tool for studying biomacromolecules and their interactions. However, quantitative detection of high-mass analytes (kDa to MDa range) remains challenging for MALDI-MS. Herein, we successfully used commercially available purified proteins (β-galactosidase and BSA) as internal standards for high-mass MALDI-MS analysis and achieved absolute quantification of several high-mass analytes. We systematically evaluated four sample deposition methods, and using the sandwich deposition method with saturated sinapinic acid as the top layer, we performed a robust quantitative analysis by high-mass MALDI-MS. Combined with chemical cross-linking, this quantitative strategy was further used to evaluate the affinity of protein-protein interactions (PPIs), specifically of two soluble protein receptors (interleukin 1 receptor and interleukin 2 receptor) and two membrane protein receptors (rhodopsin and angiotensin 2 receptor 1) with their interaction partners. The measured dissociation constants of the protein complexes formed were between 10 nM and 5 μM. We expect this high-throughput, rapid method, which does not require labeling or immobilization of any of the interaction partners, to become a viable alternative to traditional biophysical methods for studying PPIs.

High-Throughput Screening Test for Adhesion in Soft Materials Using Centrifugation.

High-throughput screening of mechanical properties can transform materials science research by both aiding in materials discovery and developing predictive models. However, only a few such assays have been reported, requiring custom or expensive equipment, while the mounting demand for enormous data sets of materials properties for predictive models is unfulfilled by the current characterization throughput. We address this problem by developing a high-throughput colorimetric adhesion screening method using a common laboratory centrifuge, multiwell plates, and microparticles. The technique uses centrifugation to apply a homogeneous mechanical detachment force across individual formulations in a multiwell plate. We also develop a high-throughput sample deposition method to prepare films with uniform thickness in each well, minimizing well-to-well variability. After establishing excellent agreement with the well-known probe tack adhesion test, we demonstrate the consistency of our method by performing the test on a multiwell plate with two different formulations in an easily discernible pattern. The throughput is limited only by the number of wells in the plates, easily reaching 103 samples/run. With its simplicity, low cost, and large dynamic range, this high-throughput method has the potential to change the landscape of adhesive material characterization.

Ultrasensitive Microstring Resonators for Solid State Thermomechanical Analysis of Small and Large Molecules.

Thermal analysis plays an important role in both industrial and fundamental research and is widely used to study thermal characteristics of a variety of materials. However, despite considerable effort using different techniques, research struggles to resolve the physicochemical nature of many thermal transitions such as amorphous relaxations or structural changes in proteins. To overcome the limitations in sensitivity of conventional techniques and to gain new insight into the thermal and mechanical properties of small- and large-molecule samples, we have developed an instrumental analysis technique using resonating low-stress silicon nitride microstrings. With a simple sample deposition method and postprocess data analysis, we are able to perform rapid thermal analysis of direct instrumental triplicate samples with only pico- to nanograms of material. Utilizing this method, we present the first measurement of amorphous alpha and beta relaxation, as well as liquid crystalline transitions and decomposition of small-molecule samples deposited onto a microstring resonator. Furthermore, sensitive measurements of the glass transition of polymers and yet unresolved thermal responses of proteins below their apparent denaturation temperature, which seem to include the true solid state glass transition of pure protein, are reported. Where applicable, thermal events detected with the setup were in good agreement with conventional techniques such as differential scanning calorimetry and dynamic mechanical analysis. The sensitive detection of even subtle thermal transitions highlights further possibilities and applications of resonating microstrings in instrumental physicochemical analysis.

Development and Characterization of a Laser-Induced Acoustic Desorption Source.

A laser-induced acoustic desorption source, developed for use at central facilities, such as free-electron lasers, is presented. It features prolonged measurement times and a fixed interaction point. A novel sample deposition method using aerosol spraying provides a uniform sample coverage and hence stable signal intensity. Utilizing strong-field ionization as a universal detection scheme, the produced molecular plume is characterized in terms of number density, spatial extend, fragmentation, temporal distribution, translational velocity, and translational temperature. The effect of desorption laser intensity on these plume properties is evaluated. While translational velocity is invariant for different desorption laser intensities, pointing to a nonthermal desorption mechanism, the translational temperature increases significantly and higher fragmentation is observed with increased desorption laser fluence.

Evaluation of sample preparation protocols for spider venom profiling by MALDI-TOF MS

Spider venoms are highly complex mixtures containing biologically active substances with potential for use in biotechnology or pharmacology. Fingerprinting of venoms by Matrix-Assisted Laser Desorption-Ionization – Time of Flight Mass Spectrometry (MALDI-TOF MS) is a thriving technology, enabling the rapid detection of peptide/protein components that can provide comparative information. In this study, we evaluated the effects of sample preparation procedures on MALDI-TOF mass spectral quality to establish a protocol providing the most reliable analytical outputs. We adopted initial sample preparation conditions from studies already published in this field. Three different MALDI matrixes, three matrix solvents, two sample deposition methods, and different acid concentrations were tested. As a model sample, venom from Brachypelma albopilosa was used. The mass spectra were evaluated on the basis of absolute and relative signal intensities, and signal resolution. By conducting three series of analyses at three weekly intervals, the reproducibility of the mass spectra were assessed as a crucial factor in the selection for optimum conditions. A sample preparation protocol based on the use of an HCCA matrix dissolved in 50% acetonitrile with 2.5% TFA deposited onto the target by the dried-droplet method was found to provide the best results in terms of information yield and repeatability. We propose that this protocol should be followed as a standard procedure, enabling the comparative assessment of MALDI-TOF MS spider venom fingerprints.

Generation of gas-phase ions from charged clusters: an important ionization step causing suppression of matrix and analyte ions in matrix-assisted laser desorption/ionization mass spectrometry.

Ionization in matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is a very complicated process. It has been reported that quaternary ammonium salts show extremely strong matrix and analyte suppression effects which cannot satisfactorily be explained by charge transfer reactions. Further investigation of the reasons causing these effects can be useful to improve our understanding of the MALDI process. The dried-droplet and modified thin-layer methods were used as sample preparation methods. In the dried-droplet method, analytes were co-crystallized with matrix, whereas in the modified thin-layer method analytes were deposited on the surface of matrix crystals. Model compounds, tetrabutylammonium iodide ([N(Bu)4 ]I), cesium iodide (CsI), trihexylamine (THA) and polyethylene glycol 600 (PEG 600), were selected as the test analytes given their ability to generate exclusively pre-formed ions, protonated ions and metal ion adducts respectively in MALDI. The strong matrix suppression effect (MSE) observed using the dried-droplet method might disappear using the modified thin-layer method, which suggests that the incorporation of analytes in matrix crystals contributes to the MSE. By depositing analytes on the matrix surface instead of incorporating in the matrix crystals, the competition for evaporation/ionization from charged matrix/analyte clusters could be weakened resulting in reduced MSE. Further supporting evidence for this inference was found by studying the analyte suppression effect using the same two sample deposition methods. By comparing differences between the mass spectra obtained via the two sample preparation methods, we present evidence suggesting that the generation of gas-phase ions from charged matrix/analyte clusters may induce significant suppression of matrix and analyte ions. The results suggest that the generation of gas-phase ions from charged matrix/analyte clusters is an important ionization step in MALDI-MS. Copyright © 2016 John Wiley & Sons, Ltd.

Comparison of Sample Preparation Methods and Evaluation of Intra- and Intersample Reproducibility in Bacteria MALDI-MS Profiling

A study is presented on the reproducibility of mass spectral profiles of the whole bacterium E. coli resulting from laser sampling at different regions within and between matrix-assisted laser desorption ionization (MALDI) samples deposited onto the plate. Samples were prepared with different deposition methods and using different MALDI matrices. The three most common matrices used in MALDI-mass spectrometry (MS) bacteria profiling, α-cyano-4-hydroxycinnamic acid (CHCA), sinapinic acid (SA), and ferulic acid (FA), were compared in this study along with two pipet-based sample deposition methods, dried-droplet and premix. Sample variability was determined by analysis of variances (ANOVA), principal component analysis (PCA), and multivariate ANOVA (MANOVA). For the two pipet-based sample deposition methods tested in this study, the intrasample variability (most commonly referred to as "spot-to-spot" reproducibility) was of the same magnitude as the intersample variability for all MALDI matrices tested. By incorporating a spray nebulizer sample deposition method to produce uniform sample/matrix mixtures onto the MALDI plate, we demonstrate that the crystalline morphology of the MALDI sample greatly influences the intrasample reproducibility (i.e., spot-to-spot) of the resulting whole cell MALDI-MS profiles. Overall, for the pipet-based deposition methods, results showed that the smallest variability in bacteria MALDI mass spectral profiles was obtained from samples deposited using the premix method, regardless of the MALDI matrix used, with the best reproducibility obtained with the CHCA matrix. It is concluded that a sample preparation strategy that reduces or eliminates the MALDI matrix morphology heterogeneity can reduce variability (i.e., spot-to-spot) of the bacteria mass spectral profiles by up to 90%.

DNA compaction by mononuclear platinum cancer drug cisplatin and the trisplatinum anticancer agent BBR3464: Differences and similarities

Cisplatin, a mononuclear platinum compound, which is known as a cancer drug for long time, can exhibit considerable side effects and is also not effective in many types of cancer. Therefore, the alternative platinum anticancer agents that can act at a much lower dose limit compared to the dose relevant for cisplatin treatment have been searched for. BBR3464, a trinuclear platinum compound, is found to exhibit cytotoxic effects at 10 to 1000 times lower dose limit, even in cisplatin-resistant cancer cells. The primary cellular target for cisplatin and BBR3464 is thought to be DNA. Herein, we report the nature of DNA structural changes that are induced by cisplatin and BBR3464, considering the same DNA sequence and similar sample deposition methods for comparison purpose. We have applied high-resolution atomic force microscopy (AFM) in order to obtain an idea about the molecular basis of BBR3464’s effectiveness at the lower dose limit. We show from the molecularly resolved AFM images that both the compounds can compact the whole dsDNA molecules, though the degree of compaction in case of BBR3464 treatment is significantly higher. Furthermore, local compaction in terms of loop structure formation could be induced by both BBR3464 and cisplatin, though BBR3464 generated microloops and macroloops both, whereas cisplatin could generate primarily the microloops. It is a significant observation that BBR3464 could induce relatively drastic DNA structural changes in terms of loop formation as well as overall DNA compaction at a molar ratio, which is 50 times less than that applied for cisplatin treatment. Implications of such structural changes in cytotoxic effects of the platinum anticancer agents will be mentioned.

Improved silica xerogel film processing for MALDI-TOF–MS quantitative analysis of peptides and small molecules

In this work, sol–gel derived silica films were prepared for direct desorption/ionization of organic compounds in MALDI-TOF–MS analysis with the aim of improving method precision and of reducing interfering signals at low m/z values. Two commonly used MALDI matrices, 2,5-dihydroxybenzoic acid (DHB) and α-cyano-4-hydroxycinnamic acid (CHCA), were incorporated into the sol–gel network in order to absorb laser energy and to induce analyte desorption/ionization with low or absent background signals in the mass spectra. To achieve a reproducible xerogel film formation, experimental parameters for its deposition were optimized. The gel matrices were characterized by Fourier Transform Infrared (FT-IR) spectroscopy, X-ray Diffraction (XRD), and Scanning Electron Microscopy (SEM) analysis. The results proved the embedding of the matrix molecules in a disperse form into the homogeneous sol–gel material. The sol–gel matrix was then tested for the qualitative and quantitative analysis of two reference peptides, such as Bradykinin and P14R. In addition, spectral quality and method performance were assessed for quantitation of melamine, a low-molecular weight compound of food safety concern. In all cases, high quality spectra and excellent mass accuracy (between 3.5 and 13 ppm) were observed. Furthermore, the experimental results evidenced a significant improvement of the measurement repeatability on spot and between spots (relative standard deviation <10%), with respect to the traditional dried-droplet sample deposition method. Good sensitivity and linearity in the concentration range explored were obtained for peptides and melamine, demonstrating the suitability of the sol–gel-based matrix to be used for quantitative analysis.

Sieve‐based device for MALDI sample preparation. III. Its power for quantitative measurements

The solid sample inhomogeneity is a weak point of traditional MALDI deposition techniques that reflects negatively on quantitative analysis. The recently developed sieve-based device (SBD) sample deposition method, based on the electrospraying of matrix/analyte solutions through a grounded sieve, allows the homogeneous deposition of microcrystals with dimensions smaller than that of the laser spot. In each microcrystal the matrix/analyte molar ratio can be considered constant. Then, by irradiating different portions of the microcrystal distribution an identical response is obtained. This result suggests the employment of SBD in the development of quantitative procedures. For this aim, mixtures of different proteins of known molarity were analyzed, showing a good relationship between molarity and intensity ratios. This behaviour was also observed in the case of proteins with quite different ionic yields. The power of the developed method for quantitative evaluation was also tested by the measurement of the abundance of IGPP[Oxi]GPP[Oxi]GLMGPP (m/z 1219) present in the collagen-α-5(IV) chain precursor, differently expressed in urines from healthy subjects and diabetic-nephropathic patients, confirming its overexpression in the presence of nephropathy. The data obtained indicate that SBD is a particularly effective method for quantitative analysis also in biological fluids of interest.

Online deposition of nano‐aerosol for matrix‐assisted laser desorption/ionization mass spectrometry

An online nano-aerosol sample deposition method for matrix-assisted laser desorption/ionization (MALDI) mass spectrometry is described in which matrix and analyte particles between 50 and 500 nm are aerodynamically focused onto a tight spot, ca. 200 microm in diameter, on the target plate under vacuum. MALDI analysis of the target is performed without additional sample preparation. The method is evaluated with insulin as the analyte and alpha-cyano-4-hydroxycinnamic acid (CHCA) as the matrix. Two preparation modes are compared with conventional dried-droplet deposition: mixture deposition where a single layer is deposited consisting of particles that contain both matrix and analyte, and layered deposition where an underlayer of matrix particles and an overlayer of analyte particles are deposited separately. Desalting is performed by adding ammonium sulfate to the solution used to generate the matrix aerosol. With mixture deposition, the optimum matrix-to-analyte mole ratio is about 500:1 compared with 5000:1 for the conventional dried-droplet method. With layered deposition, the thicknesses of the matrix and analyte layers are more important determinants of the analyte signal intensity than the matrix-to-analyte mole ratio. Analyte signal intensities are independent of matrix layer thickness above 200 nm, and the optimum analyte signal is obtained with an analyte layer thickness of about 100 nm.

Nanostructure-initiator mass spectrometry: a protocol for preparing and applying NIMS surfaces for high-sensitivity mass analysis

Nanostructure-initiator mass spectrometry (NIMS) is a new surface-based MS technique that uses a nanostructured surface to trap liquid ('initiator') compounds. Analyte materials adsorbed onto this 'clathrate' surface are subsequently released by laser irradiation for mass analysis. In this protocol, we describe the preparation of NIMS surfaces capable of producing low background and high-sensitivity mass spectrometric measurement using the initiator compound BisF17. Examples of analytes that adsorb to this surface are small molecules, drugs, lipids, carbohydrates and peptides. Typically, NIMS is used to analyze samples ranging from simple analytical standards and proteolytic digests to more complex samples such as tissues, cells and biofluids. Critical experimental considerations of NIMS are described. Specifically, NIMS sensitivity is examined as a function of pre-etch cleaning treatment, etching current density, etching time, initiator composition, sample concentration, sample deposition method and laser fluence. Typically, NIMS surface preparation can be completed in less than 2 h. Subsequent sample preparation requires 1-5 min, depending on sample deposition method. Mass spectrometric data acquisition typically takes 1-30 s per sample.

Fragmentation of Multiply-Charged Intact Protein Ions Using MALDI TOF-TOF Mass Spectrometry

Top down proteomics in a TOF-TOF instrument was further explored by examining the fragmentation of multiply charged precursors ions generated by matrix-assisted laser desorption ionization. Evaluation of sample preparation conditions allowed selection of solvent/matrix conditions and sample deposition methods to produce sufficiently abundant doubly and triply charged precursor ions for subsequent CID experiments. As previously reported, preferential cleavage was observed at sites C-terminal to acidic residues and N-terminal to proline residues for all ions examined. An increase in nonpreferential fragmentation as well as additional low mass product ions was observed in the spectra from multiply charged precursor ions providing increased sequence coverage. This enhanced fragmentation from multiply charged precursor ions became increasingly important with increasing protein molecular weight and facilitates protein identification using database searching algorithms. The useable mass range for MALDI TOF-TOF analysis of intact proteins has been expanded to 18.2 kDa using this approach.

Automated nanoliter solution deposition for total reflection X-ray fluorescence analysis of semiconductor samples

In this study, a BioDot BioJet dispensing system was investigated as a nanoliter sample deposition method for total reflection X-ray fluorescence (TXRF) analysis. The BioDot system was programmed to dispense arrays of 20 nL droplets of sample solution on Si wafers. Each 20 nL droplet was approximately 100 μm in diameter. A 10 × 10 array (100 droplets) was deposited and dried in less than 2 min at room temperature and pressure, demonstrating the efficiency of the automated deposition method. Solutions of various concentrations of Ni and Ni in different matrices were made from stock trace element standards to investigate of the effect of the matrix on the TXRF signal. The concentrations were such that the levels of TXRF signal saturation could be examined. Arrays were deposited to demonstrate the capability of drying 100 μL of vapor phase decomposition-like residue in the area of a typical TXRF detector.

The influence of electrospray deposition in matrix-assisted laser desorption/ionization mass spectrometry sample preparation for synthetic polymers.

Although electrospray sample deposition in matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) sample preparation increases the repeatability of both the MALDI signal intensity and the measured molecular mass distribution (MMD), the electrospray sample deposition method may influence the apparent MMD of a synthetic polymer. The MMDs of three polymers of differing thermal stability, polystyrene (PS), poly(ethylene glycol) (PEG), and poly(propylene glycol) (PPG), were studied by MALDI time-of-flight (TOF) MS as the electrospray deposition voltage was varied. The MMDs obtained using the electrospray deposition method were compared with those obtained for hand-spotted samples. No change was observed in the measured polymer MMD when the electrospray deposition voltage was varied in the analysis of PS, but those of PEG and PPG changed at higher electrospray voltages due to increased ion fragmentation. It was also shown that the fragmentation in the hand-spotted samples is dependent on the matrix used in sample preparation.

Quantitation of underivatized free amino acids in mammalian cell culture media using matrix assisted laser desorption ionization time-of-flight mass spectrometry.

In this investigation, a quantitative matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOFMS) method was developed for the analysis of underivatized free amino acids in mammalian cell culture media. Calibration curves were developed for 12 amino acids over the linear range of 1-100 microM with coefficients of determination ranging from r2 = 0.9220 to r2 = 0.9973. An aerospray method was utilized for the sample deposition method, and the matrix, alpha-cyano-4-hydroxycinnamic acid, served as the internal standard. This assay was used to analyze bioreactor samples from five time points in the process. Concentrations determined through interpolation of the calibration curves were comparable to those obtained via reversed-phase HPLC based analysis with an average percent difference of 19.71%. Repeatability and intermediate precision studies were also performed, and the relative standard deviations ranged from 0.5943 to 21.41 and 3.157 to 18.97, respectively.

Characterization of poly(butylene terephthalate) by size-exclusion chromatography and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Poly(butylene terephthalate) (PBT) samples have been analyzed with size-exclusion chromatography (SEC) using a mixed solvent of 1,1,1,3,3,3-hexafluoroisopropanol (HFIP) and chloroform as the mobile phase. Several matrices and different sample deposition methods have been investigated to analyze PBT with matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS). Optimum results have been acquired by depositing PBT on top of a 2,4,6-trihydroxyacetophenone matrix. The found MALDI-TOF-MS method can be used to analyze the end group functionalities of PBT, as demonstrated with the samples at hand. By combining SEC (off-line) with MALDI-TOF-MS, absolute molecular masses of PBT can be measured, and these have been found to be considerably lower than those determined with SEC using polystyrene standards.

An activated carbon substrate surface for laser desorption mass spectrometry

A method to obtain laser desorption/ionization mass spectra of organic compounds by depositing sample solutions onto a carbon substrate surface is demonstrated. The substrate consists of a thin layer of activated carbon particles immobilized on an aluminum support. In common with the porous carbon suspension samples used in previous “surface-assisted laser desorption/ionization” (SALDI) work, the mass spectra contain only a few “matrix” background ion peaks, minimizing interference with analyte ion peaks. The presence of glycerol ensured that the ion signals were stable over hundreds of laser shots. In addition, the carbon substrate surface has several advantages over the suspension samples. The use of a very thin layer of carbon significantly improves the sensitivity. Detection limits range from attomoles for crystal violet to femtomoles for bradykinin. Very little sample preparation is required as the analyte solution is simply pipetted onto the substrate surface and glycerol added. When using an alternate sample deposition method, a mass resolution for bradykinin of 1800 is achieved in linear time-of-flight mode. This is close to the resolution limit set by the detector system and above instrument specification for matrix-assisted laser desorption/ionization mass spectra.

Measurement of anisotropic behavior of dry cohesive and cohesionless powders using a cubical triaxial tester

Anisotropy of dry cohesive and cohesionless powders and the effect of particle shape and sample deposition method on the anisotropy were investigated using a cubical triaxial tester. Hydrostatic triaxial compression (HTC) tests and conventional triaxial compression (CTC) tests were conducted on four test powders: two cohesive, wheat flour (irregular-shaped particles) and potato starch (rounded); and two cohesionless, glass beads (spherical and milled glass fibers (cylindrical). The loading-unloading cycle for HTC tests was from 0-190-0 kPa. The CTC tests were conducted at 34.5 kPa confining ( σ 2 = σ 3) pressure, where σ 2 and σ 3 are intermediate and minor principal stresses, respectively. Two different deposition methods, plunging and tapping, were used to prepare cubical (50 mm) test samples. Test results showed that wheat flour and glass beads were basically isotropic materials in the stress range used in this study. Potato starch showed some anisotropy which tended to increase with applied stress. Significant anisotropy was observed for milled glass fibers (0.05 level of significance). The sample deposition method influenced the anisotropy of powder depending on the particle shape and cohesion. The anisotropy of powder was strongly dependent upon the particle shape. Cohesion was found to minimize the effect of sample deposition method. The material parameter values for the linear elastic isotropic and anisotropic constitutive equations were determined based on test data. No large difference between material parameter values was found for wheat flour, potato starch and glass beads. However, the percent difference between elastic modulus for milled glass fibers reached 37.2%, whereas for the Poisson ratio this difference was 800%.