Publications

Journal Articles

Use of High-Throughput Mass Spectrometry to Reduce False Positives in Protease uHTS Screens

Abstract

As a label-free technology, mass spectrometry (MS) enables assays to be generated that monitor the conversion of substrates with native sequences to products without the requirement for substrate modifications or indirect detection methods. Although traditional liquid chromatography (LC)-MS methods are relatively slow for a high-throughput screening (HTS) paradigm, with cycle times typically ≥ 60 s per sample, the Agilent RapidFire High-Throughput Mass Spectrometry (HTMS) System, with a cycle time of 5-7 s per sample, enables rapid analysis of compound numbers compatible with HTS. By monitoring changes in mass directly, HTMS assays can be used as a triaging tool by eliminating large numbers of false positives resulting from fluorescent compound interference or from compounds interacting with hydrophobic fluorescent dyes appended to substrates. Herein, HTMS assays were developed for multiple protease programs, including cysteine, serine, and aspartyl proteases, and applied as a confirmatory assay. The confirmation rate for each protease assay averaged <30%, independent of the primary assay technology used (i.e., luminescent, fluorescent, and time-resolved fluorescent technologies). Importantly, >99% of compounds designed to inhibit the enzymes were confirmed by the corresponding HTMS assay. Hence, HTMS is an effective tool for removing detection-based false positives from ultrahigh-throughput screening, resulting in hit lists enriched in true actives for downstream dose response titrations and hit-to-lead efforts.

 

Read full article here

Authors: Gregory C. Adam, Juncai Meng, Joseph M. Rizzo, Adam Amoss, Jeffrey W. Lusen, Amita Patel, Daniel Riley, Rachel Hunt, Paul Zuck, Eric N. Johnson, Victor N. Uebele and Jeffrey D. Hermes

Enzyme Microarrays Assembled by Acoustic Dispensing Technology

Abstract

Miniaturizing bioassays to the nanoliter scale for high-throughput screening reduces the consumption of reagents that are expensive or difficult to handle. Through the use of acoustic dispensing technology, nanodroplets containing 10 microM ATP (3 microCi/microL (32)P) and reaction buffer in 10% glycerol were positionally dispensed to the surface of glass slides to form 40-nL compartments (100 droplets/slide) for Pim1 (proviral integration site 1) kinase reactions. The reactions were activated by dispensing 4 nL of various levels of a pyridocarbazolo-cyclopentadienyl ruthenium complex Pim1 inhibitor, followed by dispensing 4 nL of a Pim1 kinase and peptide substrate solution to achieve final concentrations of 150 nM enzyme and 10 microM substrate. The microarray was incubated at 30 degrees C (97% R(h)) for 1.5 h. The spots were then blotted to phosphocellulose membranes to capture phosphorylated substrate. With phosphor imaging to quantify the washed membranes, the assay showed that, for doses of inhibitor from 0.75 to 3 microM, Pim1 was increasingly inhibited. Signal-to-background ratios were as high as 165, and average coefficients of variation for the assay were approximately 20%. Coefficients of variation for dispensing typical working buffers were under 5%. Thus, microarrays assembled by acoustic dispensing are promising as cost-effective tools that can be used in protein assay development.

 

Read full article here

Authors: E. Y. Wong and S. L. Diamond

Acoustic deposition with NIMS as a high-throughput enzyme activity assay

Abstract

Mass spectrometry (MS)-based enzyme assay has been shown to be a useful tool for screening enzymatic activities from environmental samples. Recently, reported approaches for high-specificity multiplexed characterization of enzymatic activities allow for providing detailed information on the range of enzymatic products and monitoring multiple enzymatic reactions. However, the throughput has been limited by the slow liquid-liquid handling and manual analysis. This rapid communication demonstrates the integration of acoustic sample deposition with nanostructure initiator mass spectrometry (NIMS) imaging to provide reproducible measurements of multiple enzymatic reactions at a throughput that is tenfold to 100-fold faster than conventional MS-based enzyme assay. It also provides a simple means for the visualization of multiple reactions and reaction pathways.

 

Read full article here

Authors: Matthew Greving & Xiaoliang Cheng & Wolfgang Reindl & Benjamin Bowen & Kai Deng & Katherine Louie & Michael Nyman & Joseph Cohen & Anup Singh & Blake Simmons & Paul Adams & Gary Siuzdak & Trent Northen

Nanolitre-scale crystallization using acoustic liquid-transfer technology

Abstract

Focused acoustic energy allows accurate and precise liquid transfer on scales from picolitre to microlitre volumes. This technology was applied in protein crystallization, successfully transferring a diverse set of proteins as well as hundreds of precipitant solutions from custom and commercial crystallization screens and achieving crystallization in drop volumes as small as 20 nl. Only higher concentrations (>50%) of 2-methyl-2,4-pentanediol (MPD) appeared to be systematically problematic in delivery. The acoustic technology was implemented in a workflow, successfully reproducing active crystallization systems and leading to the discovery of crystallization conditions for previously uncharacterized proteins. The technology offers compelling advantages in low-nanolitre crystallization trials by providing significant reagent savings and presenting seamless scalability for those crystals that require larger volume optimization experiments using the same vapor-diffusion format.

Read full article here

Authors: Armando G. Villasen, April
Wong, Ada Shao,  Ankur Garg,
Timothy J. Donohue, Andreas
Kuglstatterc, and Seth F. Harrisd

Screening of HIV-1 Protease Using a Combination of an Ultra-High-Throughput Fluorescent-Based Assay and RapidFire Mass Spectrometry

Abstract

HIV-1 protease (PR) represents one of the primary targets for developing antiviral agents for the treatment of HIV-infected patients. To identify novel PR inhibitors, a label-free, high-throughput mass spectrometry (HTMS) assay was developed using the RapidFire platform and applied as an orthogonal assay to confirm hits identified in a fluorescence resonance energy transfer (FRET)-based primary screen of > 1 million compounds. For substrate selection, a panel of peptide substrates derived from natural processing sites for PR was evaluated on the RapidFire platform. As a result, KVSLNFPIL, a new substrate measured to have a ~ 20- and 60-fold improvement in k cat/K m over the frequently used sequences SQNYPIVQ and SQNYPIV, respectively, was identified for the HTMS screen. About 17% of hits from the FRET-based primary screen were confirmed in the HTMS confirmatory assay including all 304 known PR inhibitors in the set, demonstrating that the HTMS assay is effective at triaging false-positives while capturing true hits. Hence, with a sampling rate of ~7 s per well, the RapidFire HTMS assay enables the high-throughput evaluation of peptide substrates and functions as an efficient tool for hits triage in the discovery of novel PR inhibitors.

Read full article here

Authors: Juncai Meng1, Ming-Tain Lai, Vandna Munshi, Jay Grobler,
John McCauley, Paul Zuck1, Eric N. Johnson, Victor N. Uebele1,
Jeffrey D. Hermes, and Gregory C. Adam

Noninvasive Fluid Property Measurements Using Acoustic Methods

Abstract

The properties of a fluid are normally determined using invasive methods. These methods may lead to possibly contaminating or consuming the sample. When only very small amounts of a valuable sample exist, noninvasive measurement methods are preferred. The properties of fluids can then be used to deduce additional properties based on known relationships. In one case, the surface tension of a fluid may be used to determine the concentration of a fluid. The authors describe a measurement technique involving excitation of the surface of the fluid and the measurement of its response. An acoustic wave is used to both excite and monitor the surface of the liquid. This technique is used to determine the concentration of DMSO and water in solution, and the result determines the amount of fluid needed to deliver an accurate amount of solute in solution.

Read full article here

Authors: Michael Forbush, Humphrey Chow, James Chiao, and Andrew Rosed

Acoustic Sample Deposition MALDI-MS (ASD-MALDI-MS): A Novel Process Flow for Quality Control Screening of Compound Libraries

Abstract

In the early stages of drug discovery, high-throughput screening (HTS) of compound libraries against pharmaceutical targets is a common method to identify potential lead molecules. For these HTS campaigns to be efficient and successful, continuous quality control of the compound collection is necessary and crucial. However, the large number of compound samples and the limited sample amount pose unique challenges. Presented here is a proof-of-concept study for a novel process flow for the quality control screening of small-molecule compound libraries that consumes only minimal amounts of samples and affords compound-specific molecular data. This process employs an acoustic sample deposition (ASD) technique for the offline sample preparation by depositing nanoliter volumes in an array format onto microscope glass slides followed by matrix-assisted laser desorption/ionization mass spectrometric (MALDI-MS) analysis. An initial study of a 384-compound array employing the ASD-MALDI-MS workflow resulted in a 75% first-pass positive identification rate with an analysis time of <1 s per sample.

Read full article here

Authors: Jefferson Chin, Elizabeth Wood, Grace S. Peters, and Dieter M. Drexler

High-Throughput RT-PCR for small-molecule screening assays

Abstract

Quantitative measurement of the levels of mRNA expression using real-time reverse transcription polymerase chain reaction (RT-PCR) has long been used for analyzing expression differences in tissue or cell lines of interest. This method has been used somewhat less frequently to measure the changes in gene expression due to perturbagens such as small molecules or siRNA. The availability of new instrumentation for liquid handling and real-time PCR analysis as well as the commercial availability of start-to-finish kits for RT-PCR has enabled the use of this method for high-throughput small-molecule screening on a scale comparable to traditional high-throughput screening (HTS) assays. This protocol focuses on the special considerations necessary for using quantitative RT-PCR as a primary small-molecule screening assay, including the different methods available for mRNA isolation and analysis.

Read full article here

Author: Joshua A. Bittker

Application Notes

A High Throughput Screening System to Identify Actionable Treatments for Cancer Patients

Notable Labs is a startup biotechnology company in San Francisco focusing on the repurposing of FDA-approved drugs to improve personalized chemotherapy. Using their Biosero WorkCell, Notable Labs identifies actionable treatments for cancer patients by screening a patients cancer cells against a vast number of drug and drug combinations (drug/drug combinations) which can then be prescribed by their physician without clinical trials. They are currently concentrating on two types of cancers, acute myeloid leukemia and glioblastoma multiforme. Prior to purchasing their WorkCell, Notable Labs needed a way to make their patient cell screening process robust, scalable and cost effective. In addition, to maximize the number of drug/drug combinations tested with the limited number of patient cells they receive, they needed to miniaturize their assays. Notable Labs collaborated with Biosero, Inc. to develop an automated cellular screening WorkCell capable of screening up to 30,000 drug/drug combinations with multiple assays against a cancer patients cells.

View application note here

Posters

A High Throughput Screening System to Identify Actionable Treatments for Cancer Patients

Our mission at Notable Labs is to identify actionable treatment protocols for cancer patients through a proprietary approach to personalized drug repurposing. We screen a vast number of combinations of FDA-approved drugs against a cancer patient’s cells to identify drug combinations that have improved ecacy in vitro and can be immediately prescribed by their doctor without a clinical trial. Our current focus is screening for patients with glioblastoma multiforme (GBM) and acute myeloid leukemia (AML). Technological advances allow Notable Labs to intelligently screen more drugs and combinations on fewer cells than before, and to interrogate larger datasets to glean clinically relevant insights for each patient. There is an increasing trend towards data-driven personalized approaches to drug repurposing, and testing drug combinations on primary or patient cells. Challenges we are focused on will improve the outcome of this approach by generating the most data possible from limited sample size, and managing variability between patient samples. By focusing on technological improvement on three aspects of drug repurposing; automated sample handling, combinatorial strategy, and direct testing on patient cells, we will build a rich, relevant reference dataset to expedite screening and implementation of cost eective treatments for eligible cancer patients. This presentation describes our rst key technological innovation: complete automation of patient sample processing and screening. Automation increases throughput, decreases cost, and improves patient data quality. Over time this strategy will generate a cost eective approach to personalized

View poster here