The adoption of mass spectrometry for high-throughput screening in drug discovery has become increasingly prevalent and has enabled label-free screening against diverse targets. Cellular assays for phenotypic screening, however, are primarily conducted by microscopy as there remain many challenges associated with conducting phenotypic screens via ultra-high throughput mass spectrometry. Following a simple on-plate extraction, infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) was employed to directly sample the cell lysate at a speed of one sample per second with high mass resolution. A549 cells were treated with compounds identified as hits in literature, including a recently reported glutaminase cellular screen. Among the test compounds were confirmed glutaminase inhibitors, proposed nuisance compounds, and cell-active but enzyme-inactive compounds. Filtered data were further processed in R for dimensionality reduction and unsupervised clustering. The general nature of dimensionality reduction enables the immediate use of this method in applications other than glutaminase inhibition. Though we observed that all compounds affected the intracellular conversion of glutamine to glutamate, there were clear metabolic differences between the biochemically active compounds and the off-target false hits. Moreover, two nuisance compounds were observed to cluster separately from the confirmed glutaminase inhibitors in the observed metabolite fingerprints. This proof-of-concept work establishes a workflow that enables high-throughput mass spectrometry-based phenotypic screening. The methods proposed herein, at the throughput enabled by IR-MALDESI, could offer a new avenue for the discovery of novel drugs.

High-throughput experimentation enables rapid reaction optimization by leveraging reaction miniaturization, automated processes, and advanced high-throughput analytical methods. To more effectively apply these principles to reactions mediated by photoredox catalysis, we developed the photoredox optimization (PRO) reactor. PRO is an automated platform that provides precise control over the delivered light irradiance to optically thin, temperature-controlled reaction volumes. Combined with high-intensity laser illumination, PRO facilitates accelerated photoredox reaction scouting using <10 mu L of reaction material. Crude products from PRO reactions are automatically transferred to microplates for analysis by infrared matrix-assisted laser desorption electrospray ionization mass spectrometry (IR-MALDESI-MS) which can quantify 384 reactions in under 6 minutes. Validation of the PRO reactor was achieved through a series of challenging decarboxylative cross-coupling reactions, which resulted in improved isolated yields up to 58%. PRO then enabled the design and execution of higher throughput 384-reaction HTE arrays which achieved improved yields for two previously unsuccessful photoredox cross-couplings, ultimately identifying novel reaction conditions outside the scope of our traditional 96-reaction arrays.

Native mass spectrometry (MS) is a powerful analytical technique to directly probe noncovalent protein-protein and protein-ligand interactions. However, not every MS platform can preserve proteins in their native conformation due to high energy deposition from the utilized ionization source. Most small molecules approved as drugs and in development interact with their targets through noncovalent interactions. Therefore, rapid methods to analyze noncovalent protein-ligand interactions are necessary for the early stages of the drug discovery pipeline. Herein, we describe a method for analyzing noncovalent protein-ligand complexes by IR-MALDESI-MS with analysis times of ∼13 s per sample. Carbonic anhydrase and the kinase domain of Bruton's tyrosine kinase are paired with known noncovalent binders to evaluate the effectiveness of native MS by IR-MALDESI.

Over the last 5 years, IR-MALDESI-MS (Infrared Matrix-Assisted Laser Desorption Electrospray Ionization Mass Spectrometry) has been demonstrated for use in a range of high-throughput biochemical and cellular assays with remarkable sample acquisition rates up to 22 Hz for a single 384-well assay plate. With such high single plate acquisition rates, the rate limiting step becomes how fast subsequent plates can be presented to the MS for analysis. To make this transfer as fast as possible while maintaining safe operation in a laboratory environment, we developed a collaborative robotic plate transfer system (CRPTS) that combines a 6-axis robot with dual plate grippers, a 7th axis conveyor stage, and a 420-plate capacity sample loading window. As a demonstration of the throughput and flexibility of CRPTS, we performed a biochemical assay that monitored the oxidation of tris(2-carboxyethyl)phosphine (TCEP) to screen for nuisance compounds. Using continuous and step motion scan profiles, we analyzed 158,799 compounds contained in 448 assay plates over the course of 12.5 h (Z-Factor=0.87) and 17.5 h (Z-factor=0.99), respectively. Extrapolating these results enables the screening of a million compounds within 6-7 working days.

Cellular pharmacodynamic assays are crucial aspects of lead optimization programs in drug discovery. These assays are sometimes difficult to develop, oftentimes distal from the target and frequently low throughput, which necessitates their incorporation in the drug discovery funnel later than desired. The earlier direct pharmacodynamic modulation of a target can be established, the fewer resources are wasted on compounds that are acting via an off-target mechanism. Mass spectrometry is a versatile tool that is often used for direct, proximal cellular pharmacodynamic assay analysis, but liquid chromatography-mass spectrometry methods are low throughput and are unable to fully support structure-activity relationship efforts in early medicinal chemistry programs. Infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) is an ambient ionization method amenable to high-throughput cellular assays, capable of diverse analyte detection, ambient and rapid laser sampling processes, and low cross-contamination. Here, we demonstrate the capability of IR-MALDESI for the detection of diverse analytes directly from cells and report the development of a high-throughput, label-free, proximal cellular pharmacodynamic assay using IR-MALDESI for the discovery of glutaminase inhibitors and a biochemical assay for hit confirmation. We demonstrate the throughput with a ∼100,000-compound cellular screen. Hits from the screening were confirmed by retesting in dose-response with mass spectrometry-based cellular and biochemical assays. A similar workflow can be applied to other targets with minimal modifications, which will speed up the discovery of cell active lead series and minimize wasted chemistry resources on off-target mechanisms.

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.

ABSTRACT The adoption of mass spectrometry for high-throughput screening in drug discovery has become increasingly prevalent and has enabled label-free screening against diverse targets. Cellular assays for phenotypic screening, however, are primarily conducted by microscopy as there remain many challenges associated with conducting phenotypic screens via ultra-high throughput mass spectrometry. Following a simple on-plate extraction, infrared matrix assisted laser desorption electrospray ionization (IR-MALDESI) was employed to directly sample the cell lysate at a speed of one second per sample. A549 cells were treated with compounds identified as hits in literature including a recently reported glutaminase cellular screen. Among the test compounds, there were confirmed glutaminase inhibitors, proposed nuisance compounds, and cell-active but enzyme-inactive compounds. Filtered data were further processed in R for dimensionality reduction and unsupervised clustering. Though it was observed that all compounds affected the intracellular conversion of glutamine to glutamate, there were clear metabolic differences between the biochemically active compounds and the off-target hits. Moreover, two nuisance compounds were observed to cluster separately from the confirmed glutaminase inhibitors in the observed metabolite fingerprints. This proof-of-concept work establishes a complete workflow that enables highthroughput mass spectrometry-based phenotypic screening. The methods proposed herein, at the throughput enabled by IR-MALDESI, could offer a new avenue for discovery of novel drugs.

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.

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.

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) is a hybrid, ambient ionization source that combines the advantages of electrospray ionization and matrix-assisted laser desorption/ionization, making it a versatile tool for both high-throughput screening (HTS) and mass spectrometry imaging (MSI) studies. To expand the capabilities of the IR-MALDESI source, an entirely new architecture was designed to overcome the key limitations of the previous source. This next-generation (NextGen) IR-MALDESI source features a vertically mounted IR-laser, a planar translation stage with computerized sample height control, an aluminum enclosure, and a novel mass spectrometer interface plate. The NextGen IR-MALDESI source has improved user-friendliness, improved overall versatility, and can be coupled to numerous Orbitrap mass spectrometers to accommodate more research laboratories. In this work, we highlight the benefits of the NextGen IR-MALDESI source as an improved platform for MSI and direct analysis. We also optimize the NextGen MALDESI source component geometries to increase target ion abundances over a wide m/z range. Finally, documentation is provided for each NextGen IR-MALDESI part so that it can be replicated and incorporated into any lab space.

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.

While there are a plethora of in vivo fiber-optic spectroscopic techniques that have demonstrated the ability to detect a number of diseases in research trials with highly trained personnel familiar with the operation of experimental optical technologies, very few techniques show the same level of success in large multicenter trials. To meet the stringent requirements for a viable optical spectroscopy system to be used in a clinical setting, we developed components including an automated calibration tool, optical contact sensor for signal acquisition, and a methodology for real-time in vivo probe calibration correction. The end result is a state-of-the-art medical device that can be realistically used by a physician with spectroscopic fiber-optic probes. We show how the features of this system allow it to have excellent stability measuring two scattering phantoms in a clinical setting by clinical staff with similar to 0.5% standard deviation over 25 unique measurements on different days. In addition, we show the systems' ability to overcome many technical obstacles that spectroscopy applications often face such as speckle noise and user variability. While this system has been designed and optimized for our specific application, the system and design concepts are applicable to most in vivo fiber-optic-based spectroscopic techniques. (C)The Authors. Published by SPIE under a Creative Commons Attribution 3.0 Unported License.

Diverging beam illumination is widely used in many optical techniques especially in fiber optic applications and coherence phenomenon is one of the most important properties to consider for these applications. Until now, people have used Monte Carlo simulations to study the backscattering coherence phenomenon in collimated beam illumination only. We are the first one to study the coherence phenomenon under the exact diverging beam geometry by taking into account the impossibility of the existence for the exact time-reversed path pairs of photons, which is the main contribution to the backscattering coherence pattern in collimated beam. In this work, we present a Monte Carlo simulation that considers the influence of the illumination numerical aperture. The simulation tracks the electric field for the unique paths of forward path and reverse path in time-reversed pairs of photons as well as the same path shared by them. With this approach, we can model the coherence pattern formed between the pairs by considering their phase difference at the collection plane directly. To validate this model, we use the Low-coherence Enhanced Backscattering Spectroscopy, one of the instruments looking at the coherence pattern using diverging beam illumination, as the benchmark to compare with. In the end, we show how this diverging configuration would significantly change the coherent pattern under coherent light source and incoherent light source. This Monte Carlo model we developed can be used to study the backscattering phenomenon in both coherence and non-coherence situation with both collimated beam and diverging beam setups.

blot in GC cell lines and primary tumors.The functional role of TEAD1/4 in GC was demonstrated by MTT proliferation assays, monolayer colony formation, cell invasion and migration, cell cycle analysis, and in vivo study by siRNA-mediated knockdown.The clinicopathological correlation of TEAD1/4 in GC was achieved by immunohistochemistry of tissue microarray.The prediction of miRNAs which potentially target TEAD1/4 was performed by TargetScan (http://www.targetscan.org/)and miRDB (http://mirdb.org).miR-377-3p, miR-1343-3p and miR-4269 were screened out for further validation.The miRNA regulation on TEAD1/4 was confirmed by qRT-PCR, Western blot and dual luciferase activity assays by ectopic expression of these three miRNAs.The expression correlation of TEAD1/4 and miRNAs was evaluated in 28 primary tumors.Results: TEAD1/4 is overexpressed in GC cell lines and primary tumors compared with normal gastric epithelium.Its knockdown exhibited significant anti-oncogenic effect both in vitro and in vivo.Downregulation of TEAD1/ 4 suppressed cell proliferation, reduced more than 45% monolayer colony formation, and inhibited at least 33% of the ability of cell invasion and migration.The upregulated TEAD1/ 4 in GC mainly localized in the nuclear and it overexpression correlates with unfavorable outcome.TEAD1 was confirmed to be a direct target of miR-377-3p and miR-4269, and TEAD4 was found to be negatively regulated by miR-1343-3p and miR-4269 in GC.Enforced overexpression of these three miRNAs in GC cells also exerts tumor suppressor function.miRNA expression analysis revealed the downregulation of miR-377-3p, miR-1343-3p and miR-4269 GCs when compared with adjacent normal epithelium samples.And the expression of miRNAs and TEAD1/4 shows negative correlation in primary GC samples.Conclusion: In conclusion, the activation of TEAD1/4 in GC is partly due to the downregulation of tumor suppressor miRNAs, miR-377, miR-1343 and miR-4269.These findings create clinical implications that targeting TEAD1/4 by miRNAs might have therapeutic potential in GC.

One of the major challenges in population screening is identifying the patients at risk of cancer. For instance, in colorectal cancer, while the entire population of age 50 should be subject to screening, the lifetime risk of CRC is only ~5%. Indeed, average risk colonoscopy can detect significant polyps <10% of the time. Screening for low prevalence lesions leads to increase costs, unnecessary morbidity and most particularly false positives. Thus, risk stratification to identify patients most likely to benefit from tests such as colonoscopy is critical.

We present an ultra-simple miniature fiber optic probe to measure spatially and spectrally resolved diffuse reflectance in the sub-diffuse regime (i.e. measurements with source-detector separation less than a transport mean free path) in-vivo. This probe has a robust and simple design with a small footprint (<.5 mm diameter). We show that our probe has sensitivity to structures scattering light an order of magnitude smaller than the diffraction limit, and thus can be used to quantify alterations in the very smallest structures in tissue (e.g. organelles, chromatin, collagen fibers, etc.). Specifically, the probe samples the spatial profile of diffuse reflectance in the sub-diffusion regime (P(r), r<<1 mm). P(r) can be used to quantify the entire shape of the phase function, F(θ). The shape of the refractive index correlation function Bn(rd) (through which the spatial distribution of mass is defined) can be analytically derived from the shape of F(θ) through application of the Born approximation. Therefor measurements of P(r) can elucidate F(θ) and Bn(rd). This ability has tremendous potential for use as a diagnostic tool and broad applications for probing the nanoscale environment of tissue in-vivo.

While there are a plethora of in-vivo spectroscopic techniques that have demonstrated the ability to detect a number of diseases in research trials, very few techniques have successfully become a fully realized clinical technology. This is primarily due to the stringent demands on a clinical device for widespread implementation. Some of these demands include: simple operation requiring minimal or no training, safe for in-vivo patient use, no disruption to normal clinic workflow, tracking of system performance, warning for measurement abnormality, and meeting all FDA guidelines for medical use. Previously, our group developed a fiber optic probe-based optical sensing technique known as low-coherence enhanced backscattering spectroscopy (LEBS) to quantify tissue ultrastructure in-vivo. Now we have developed this technique for the application of prescreening patients for colonoscopy in a primary care (PC) clinical setting. To meet the stringent requirements for a viable medical device used in a PC clinical setting, we developed several novel components including an automated calibration tool, optical contact sensor for signal acquisition, and a contamination sensor to identify measurements which have been affected by debris. The end result is a state-of-the-art medical device that can be realistically used by a PC physician to assess a person’s risk for harboring colorectal precancerous lesions. The pilot study of this system shows great promise with excellent stability and accuracy in identifying high-risk patients. While this system has been designed and optimized for our specific application, the system and design concepts are universal to most in-vivo fiber optic based spectroscopic techniques.

Alterations in high order chromatin, with concomitant modulation in gene expression, are one of the earliest events in the development of colorectal cancer. Cohesins are a family of proteins that modulate high-order chromatin, although the role in colorectal cancer remains incompletely understood. We, therefore, assessed the role of cohesin SA1 in colorectal cancer biology and as a biomarker focusing in particular on the increased incidence/mortality of colorectal cancer among African-Americans. Immunohistochemistry on tissue arrays revealed dramatically decreased SA1 expression in both adenomas (62%; P = 0.001) and adenocarcinomas (75%; P = 0.0001). RT-PCR performed in endoscopically normal rectal biopsies (n = 78) revealed a profound decrease in SA1 expression in adenoma-harboring patients (field carcinogenesis) compared with those who were neoplasia-free (47%; P = 0.03). From a racial perspective, colorectal cancer tissues from Caucasians had 56% higher SA1 expression than in African-Americans. This was mirrored in field carcinogenesis where healthy Caucasians expressed more SA1 at baseline compared with matched African-American subjects (73%; P = 0.003). However, as a biomarker for colorectal cancer risk, the diagnostic performance as assessed by area under ROC curve was greater in African-Americans (AUROC = 0.724) than in Caucasians (AUROC = 0.585). From a biologic perspective, SA1 modulation of high-order chromatin was demonstrated with both biophotonic (nanocytology) and chromatin accessibility [micrococcal nuclease (MNase)] assays in SA1-knockdown HT29 colorectal cancer cells. The functional consequences were underscored by increased proliferation (WST-1; P = 0.0002, colony formation; P = 0.001) in the SA1-knockdown HT29 cells. These results provide the first evidence indicating a tumor suppressor role of SA1 in early colon carcinogenesis and as a risk stratification biomarker giving potential insights into biologic basis of racial disparities in colorectal cancer. Cancer Prev Res; 9(11); 844–54. ©2016 AACR.

At the forefront of ecosystems adversely affected by climate change, coral reefs are sensitive to anomalously high temperatures which disassociate (bleaching) photosynthetic symbionts (Symbiodinium) from coral hosts and cause increasingly frequent and severe mass mortality events. Susceptibility to bleaching and mortality is variable among corals, and is determined by unknown proportions of environmental history and the synergy of Symbiodinium- and coral-specific properties. Symbiodinium live within host tissues overlaying the coral skeleton, which increases light availability through multiple light-scattering, forming one of the most efficient biological collectors of solar radiation. Light-transport in the upper ~200 μm layer of corals skeletons (measured as ‘microscopic’ reduced-scattering coefficient, \( \mu ^{\prime}_{{S,m}} \)), has been identified as a determinant of excess light increase during bleaching and is therefore a potential determinant of the differential rate and severity of bleaching response among coral species.