Mass spectrometry (MS) is the primary analytical tool used to characterize proteins within the biopharmaceutical industry. Electrospray ionization (ESI) coupled to liquid chromatography (LC) is the ...current gold standard for intact protein analysis. However, inherent speed limitations of LC/MS prevent analysis of large sample numbers (>1000) in a day. Infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI-MS), an ambient ionization MS technology, has recently been established as a platform for high-throughput small molecule analysis. Here, we report the applications of such a system for the analysis of intact proteins commonly performed within the drug discovery process. A wide molecular weight range of proteins 10–150 kDa was detected on the system with improved tolerance to salts and buffers compared to ESI. With high concentrations and model proteins, a sample rate of up to 22 Hz was obtained. For proteins at low concentrations and in buffers used in commonly employed assays, robust data at a sample rate of 1.5 Hz were achieved, which is ∼22× faster than current technologies used for high-throughput ESI-MS-based protein assays. In addition, two multiplexed plate-based high-throughput sample cleanup methods were coupled to IR-MALDESI-MS to enable analysis of samples containing excessive amounts of salts and buffers without fully compromising productivity. Example experiments, which leverage the speed of the IR-MALDESI-MS system to monitor NISTmAb reduction, protein autophosphorylation, and compound binding kinetics in near real time, are demonstrated.
Modeling of coherent polarized light propagation in turbid scattering medium by the Monte Carlo method provides an ultimate understanding of coherent effects of multiple scattering, such as ...enhancement of coherent backscattering and peculiarities of laser speckle formation in dynamic light scattering (DLS) and optical coherence tomography (OCT) diagnostic modalities. In this report, we consider two major ways of modeling the coherent polarized light propagation in scattering tissue-like turbid media. The first approach is based on tracking transformations of the electric field along the ray propagation. The second one is developed in analogy to the iterative procedure of the solution of the Bethe-Salpeter equation. To achieve a higher accuracy in the results and to speed up the modeling, both codes utilize the implementation of parallel computing on NVIDIA Graphics Processing Units (GPUs) with Compute Unified Device Architecture (CUDA). We compare these two approaches through simulations of the enhancement of coherent backscattering of polarized light and evaluate the accuracy of each technique with the results of a known analytical solution. The advantages and disadvantages of each computational approach and their further developments are discussed. Both codes are available online and are ready for immediate use or download.
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•Prospective small molecule drugs can precipitate when injected into blood at elevated concentrations for toxicology studies.•Improved in-vitro solubility measurements in a flowing ...system are needed to better understand in-vivo precipitation.•OSPREY is a novel in-vitro flow-through system that quantifies solubility in whole blood.
Intravenous (IV) administration of poorly water-soluble small molecule therapeutics can lead to precipitation during mixing with blood. This can limit characterization of pharmacological and safety endpoints in preclinical models. Most often, tests of kinetic and thermodynamic solubility are used to optimize the formulation for solubility prior to infusion in animals, but these do not capture the dynamic precipitation processes that take place during in-vivo administration. To better capture the fluid dynamic processes that occur during IV administration, we developed the Optical Spatial PREcipitation analYzer (OSPREY) as a method to quantify the amount and size of compound precipitates in whole blood using a flow-through system that mimics IV administration. Here, we describe the OSPREY device and its underlying imaging processing methods. We then validate the ability to accurately segment particles according to their size using monodisperse suspensions of microspheres (diameter 50 to 425 µm). Next, we use a tool compound, ABT-737, to study the effects of compound concentration, vessel flow rate, compound infusion rate and vessel diameter on precipitation. Finally, we use the physiological diameter and flow rate of rat femoral vein and dog saphenous vein to demonstrate the potential of OSPREY to model in-vivo precipitation in a controlled, dynamic in-vitro assay.
Mass spectrometry (MS) can provide high sensitivity and specificity for biochemical assays without the requirement of labels, eliminating the risk of assay interference. However, its use had been ...limited to lower-throughput assays due to the need for chromatography to overcome ion suppression from the sample matrix. Direct analysis without chromatography has the potential for high throughput if sensitivity is sufficient despite the presence of a matrix. Here, we report and demonstrate a novel direct analysis high-throughput MS system based on infrared matrix-assisted desorption electrospray ionization (IR-MALDESI) that has a potential acquisition rate of 33 spectra/s. We show the development of biochemical assays in standard buffers for wild-type isocitrate dehydrogenase 1 (IDH1), diacylglycerol kinase zeta (DGKζ), and p300 histone acetyltransferase (P300) to demonstrate the suitability of this system for a broad range of high-throughput lead discovery assays. A proof-of-concept pilot screen of ∼3k compounds is also shown for IDH1 and compared to a previously reported fluorescence-based assay. We were able to obtain reliable data at a speed amenable for high-throughput screening of large-scale compound libraries.
Optical interactions with biological tissue provide powerful tools for study, diagnosis, and treatment of disease. When optical methods are used in applications involving tissue, scattering of light ...is an important phenomenon. In imaging modalities, scattering provides contrast, but also limits imaging depth, so models help optimize an imaging technique. Scattering can also be used to collect information about the tissue itself providing diagnostic value. Therapies involving focused beams require scattering models to assess dose distribution. In all cases, models of light scattering in tissue are crucial to correctly interpreting the measured signal. Here, we review a versatile model of light scattering that uses the Whittle-Matérn correlation family to describe the refractive index correlation function Bn(rd). In weakly scattering media such as tissue, Bn(rd) determines the shape of the power spectral density from which all other scattering characteristics are derived. This model encompasses many forms such as mass fractal and the Henyey-Greenstein function as special cases. We discuss normalization and calculation of optical properties including the scattering coefficient and anisotropy factor. Experimental methods using the model are also described to quantify tissue properties that depend on length scales of only a few tens of nanometers.
Lung cancer remains the leading cause of cancer deaths in the US with >150,000 deaths per year. In order to more effectively reduce lung cancer mortality, more sophisticated screening paradigms are ...needed. Previously, our group demonstrated the use of low-coherence enhanced backscattering (LEBS) spectroscopy to detect and quantify the micro/nano-architectural correlates of colorectal and pancreatic field carcinogenesis. In the lung, the buccal (cheek) mucosa has been suggested as an excellent surrogate site in the "field of injury". We, therefore, wanted to assess whether LEBS could similarly sense the presence of lung. To this end, we applied a fiber-optic LEBS probe to a dataset of 27 smokers without diagnosed lung cancer (controls) and 46 with lung cancer (cases), which was divided into a training and a blinded validation set (32 and 41 subjects, respectively). LEBS readings of the buccal mucosa were taken from the oral cavity applying gentle contact. The diagnostic LEBS marker was notably altered in patients harboring lung cancer compared to smoking controls. The prediction rule developed on training set data provided excellent diagnostics with 94% sensitivity, 80% specificity, and 95% accuracy. Applying the same threshold to the blinded validation set yielded 79% sensitivity and 83% specificity. These results were not confounded by patient demographics or impacted by cancer type or location. Moreover, the prediction rule was robust across all stages of cancer including stage I. We envision the use of LEBS as the first part of a two-step paradigm shift in lung cancer screening in which patients with high LEBS risk markers are funnelled into more invasive screening for confirmation.
Astrocytes are increasingly believed to play an important role in neurovascular coupling. Recent in vivo studies have shown that intracellular calcium levels in astrocytes correlate with reactivity ...in adjacent diving arterioles. However, the hemodynamic response to stimulation involves a complex orchestration of vessel dilations and constrictions that spread rapidly over wide distances. In this work, we study the three-dimensional cytoarchitecture of astrocytes and their interrelations with blood vessels down through layer IV of the mouse somatosensory cortex using in vivo two-photon microscopy. Vessels and astrocytes were visualized through intravenous dextran-conjugated fluorescein and cortically applied sulforhodamine 101 (SR101), respectively. In addition to exploring astrocyte density, vascular proximity, and microvascular density, we found that sheathing of subpial vessels by astrocyte processes was continuous along all capillaries, arterioles, and veins, comprising a highly interconnected pathway through which signals could feasibly be relayed over long distances via gap junctions. An inner SR101-positive sheath noted along pial and diving arterioles was determined to be nonastrocytic, and appears to represent selective SR101 staining of arterial endothelial cells. Our findings underscore the intimate relationship between astrocytes and all cortical blood vessels, and suggest that astrocytes could influence neurovascular regulation at a range of sites, including the capillary bed and pial arterioles.
Which range of structures contributes to light scattering in a continuous random media, such as biological tissue? In this Letter, we present a model to study the structural length-scale sensitivity ...of scattering in continuous random media under the Born approximation. The scattering coefficient μs, backscattering coefficient μb, anisotropy factor g, and reduced scattering coefficient μs* as well as the shape of the spatial reflectance profile are calculated under this model. For media with a biologically relevant Henyey-Greenstein phase function with g∼0.93 at wavelength λ=633 nm, we report that μs* is sensitive to structural length-scales from 46.9 nm to 2.07 μm (i.e., λ/13 to 3λ), μb is sensitive from 26.7 to 320 nm (i.e., λ/24 to λ/2), and the spatial reflectance profile is sensitive from 30.8 nm to 2.71 μm (i.e., λ/21 to 4λ).
Deconvolution from intact protein mass-to-charge spectra to mass spectra is essential to generate interpretable data for mass spectrometry (MS) platforms coupled to ionization sources that produce ...multiply charged species. Infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) can be used to analyze intact proteins in multiwell microtiter plates with speed matching small molecule analyses (at least 1 Hz). However, the lack of compatible deconvolution software has limited its use in high-throughput screening applications. Most existing automated deconvolution software packages work best for data generated from LC–MS, and to the best of our knowledge, there is no software capable of performing fast plate-based mass spectral deconvolution. Herein we present the use of a new workflow in ProSight Native for the deconvolution of protein spectra from entire well plates that can be completed within 3 s. First, we successfully demonstrated the potential increased throughput benefits produced by the combined IR-MALDESI-MS - ProSight Native platform using protein standards. We then conducted a screen for Bruton’s tyrosine kinase (BTK) covalent binders against a well-annotated compound collection consisting of 2232 compounds and applied ProSight Native to deconvolute the protein spectra. Seventeen hits including five known BTK covalent inhibitors in the compound set were identified. By alleviating the data processing bottleneck using ProSight Native, it may be feasible to analyze and report covalent screening results for >200,000 samples in a single day.
Infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) mass spectrometry is an ambient-direct sampling method that is being developed for high-throughput, label-free, ...biochemical screening of large-scale compound libraries. Here, we report the development of an ultra-high-throughput continuous motion IR-MALDESI sampling approach capable of acquiring data at rates up to 22.7 samples per second in a 384-well microtiter plate. At top speed, less than 1% analyte carryover is observed from well-to-well, and signal intensity relative standard deviations (RSD) of 11.5% and 20.9% for 3 μM 1-hydroxymidazolam and 12 μM dextrorphan, respectively, are achieved. The ability to perform parallel kinetics studies on 384 samples with a ∼30 s time resolution using an isocitrate dehydrogenase 1 (IDH1) enzyme assay is shown. Finally, we demonstrate the repeatability and throughput of our approach by measuring 115200 samples from 300 microtiter plate reads consecutively over 5.54 h with RSDs under 8.14% for each freshly introduced plate. Taken together, these results demonstrate the use of IR-MALDESI at sample acquisition rates that surpass other currently reported direct sampling mass spectrometry approaches used for high-throughput compound screening.
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