Meeting Report on the 3rd Chinese American Society for Mass Spectrometry Conference-Advancing Biological and Pharmaceutical Mass Spectrometry.

Following the highly successful Chinese American Society for Mass Spectrometry (CASMS) conferences in the previous 2 years, the 3rd CASMS Conference was held virtually on August 28-31, 2023, using the Gather.Town platform to bring together scientists in the MS field. The conference offered a 4-day agenda with a scientific program consisting of two plenary lectures, and 14 parallel symposia in which a total of 70 speakers presented technological innovations and their applications in proteomics and biological MS and metabo-lipidomics and pharmaceutical MS. In addition, 16 invited speakers/panelists presented at two research-focused and three career development workshops. Moreover, 86 posters, 12 lightning talks, 3 sponsored workshops, and 11 exhibitions were presented, from which 9 poster awards and 2 lightning talk awards were selected. Furthermore, the conference featured four young investigator awardees to highlight early-career achievements in MS from our society. The conference provided a unique scientific platform for young scientists (i.e. graduate students, postdocs, and junior faculty/investigators) to present their research, meet with prominent scientists, learn about career development, and job opportunities (http://casms.org).

Development of a Predictive Multiple Reaction Monitoring (MRM) Model for High-Throughput ADME Analyses Using Learning-to-Rank (LTR) Techniques.

Multiple Reaction Monitoring (MRM) is an important MS/MS technique commonly used in drug discovery and development, allowing for the selective and sensitive quantification of compounds in complex matrices. However, compound optimization can be resource intensive and requires experimental determination of product ions for each compound. In this study, we developed a Learning-to-Rank (LTR) model to predict the product ions directly from compound structures, eliminating the requirement for MRM optimization experiments. Experimentally determined MRM conditions for 5757 compounds were used to develop the model. Using the MassChemSite software, theoretical fragments and their mass-to-charge ratios were generated, which were then matched to the experimental product ions to create a data set. Each possible fragment was ranked based on its intensity in the experimental data. Different LTR models were built on a training split. Hyperparameter selection was performed using 5-fold cross validation. The models were evaluated using the Normalized Discounted Cumulative Gain at top k (NDCG@k) and the Coverage at top k (Coverage@k) metrics. Finally, the model was applied to predict MRM conditions for a prospective set of 235 compounds in high-throughput Caco-2 permeability and metabolic stability assays, and quantification results were compared to those obtained with experimentally acquired MRM conditions. The LTR model achieved a NDCG@5 of 0.732 and Coverage@5 of 0.841 on the validation split, and its predictions led to 97% of biologically equivalent results in the Caco-2 permeability and metabolic stability assays.

Meeting Report on the 2nd Chinese American Society for Mass Spectrometry Conference: Advancing Biological and Pharmaceutical Mass Spectrometry.

The 2nd CASMS conference was held virtually through Gather. Town platform from October 17 to 21, 2022, with a total of 363 registrants including an outstanding and diverse group of scientists at the forefront of their research fields from both academia and industry worldwide, especially in the United States and China. The conference offered a 5-day agenda with an exciting scientific program consisting of two plenary lectures, 14 parallel symposia, and 4 special sessions in which a total of 97 invited speakers presented technological innovations and their applications in proteomics & biological mass spectrometry and metabo-lipidomics & pharmaceutical mass spectrometry. In addition, 18 invited speakers/panelists presented at 3 research-focused and 2 career development workshops. Moreover, 144 posters, 54 lightning talks, 5 sponsored workshops, and 14 exhibitions were presented, from which 20 posters and 8 lightning talks received presentation awards. Furthermore, the conference featured 1 MCP lectureship and 5 young investigator awardees for the first time to highlight outstanding mid-career and early-career rising stars in mass spectrometry from our society. The conference provided a unique scientific platform for young scientists (i.e., graduate students, postdocs and junior faculty/investigators) to present their research, meet with prominent scientists, and learn about career development and job opportunities (http://casms.org).

Comprehensive characterization and optimization of Caco-2 cells enabled the development of a miniaturized 96-well permeability assay.

Assessment of compound permeability through a Caco-2 cell monolayer is a well-accepted model to evaluate its in-vivo permeability potential and transporter interaction. While this assay has commonly been conducted using a 24-well assay plate format, a miniaturised 96-well assay format is highly desirable to achieve greater capacity and higher efficiency.Previous attempts to convert this assay from 24-well to 96-well format at our lab, however, had met with varied efflux capacities and unacceptable efflux ratios for digoxin, a substrate of P-glycoprotein (Pgp), which indicated inadequate Pgp transporter expression in the 96-well format.These challenges in converting the assays were attributed to the heterogeneous and unstable nature of the Caco-2 cells. To overcome the challenges, single-cell sorting of Caco-2 cells was conducted by flow cytometry to obtain a more homogeneous and stable cell population. The sorted cells were then seeded to 96-well transwell plates and the Pgp expression under various cell culture conditions was monitored by a LC-MS/MS-based targeted proteomics method.Through cell sorting and direct Pgp expression measurement, Caco-2 cells with adequate and sustained Pgp expression in a 96-well format were obtained, which led to the successful development and implementation of a 96-well Caco-2 assay with significant efficiency gain and faster turnaround time than the historical 24-well assay.

Rapid Compound Integrity Assessment for High-Throughput Screening Hit Triaging.

Hits from high-throughput screening (HTS) assays are typically evaluated using cheminformatics and/or empirical approaches before a decision for follow-up (activity confirmation and/or sample resynthesis) is made. However, the compound integrity (i.e., identity and purity) of these hits often remains largely unknown at this stage, since many compounds in the screening collection could undergo various changes such as degradation, polymerization, and precipitation during storage over time. When compound integrity is actually assessed for HTS hits postassay to address this issue, the process often increases the overall cycle time by weeks due to the reacquisition of the samples and the lengthy liquid chromatography-ultraviolet/mass spectrometric analysis time. Here we present a novel approach where compound integrity data are collected concurrently with the concentration-response curve (CRC) stage of HTS, with both assays occurring either in parallel on two distributions from the same liquid sample or serially using the original source liquid sample. The rapid generation of compound integrity data has been enabled by a high-speed ultra-high-pressure liquid chromatography-ultraviolet/mass spectrometric platform capable of analyzing ~2000 samples per instrument per week. From this parallel approach, both compound integrity and CRC potency results for screening hits become available to medicinal chemists at the same time, which has greatly enhanced the decision-making process for hit follow-up and progression. In addition, the compound integrity results from recent hits provide a real-time and representative "snapshot" of the sample integrity of the entire compound collection, and the data can be used for in-depth analyses of the screening collection.

Acoustic Ejection/Full-Scan Mass Spectrometry Analysis for High-Throughput Compound Quality Control.

High-throughput analysis of compound dissolved in DMSO and arrayed in multiwell plates for quality control (QC) purposes has widespread utility in drug discovery, ranging from the QC of assay-ready plates dispatched by compound management, to compound integrity check in the screening collection, to reaction monitoring of chemical syntheses in microtiter plates. Due to the large number of samples (thousands per batch) involved, these workflows can put a significant burden on the liquid chromatography-mass spectrometry (LC-MS) platform typically used. To achieve the required speed of seconds per sample, several chromatography-free MS approaches have previously been used with mixed results. In this study, we demonstrated the feasibility of acoustic ejection-mass spectrometry (AE-MS) in full-scan mode for high-throughput compound QC in miniaturized formats, featuring direct, contactless liquid sampling, minimal sample consumption, and ultrafast analytical speed. The sample consumption and analysis time by AE-MS represent, respectively, a 1000-fold and 30-fold reduction compared with LC-MS. In qualitative QC, AE-MS generated comparable results to conventional LC-MS in identifying the presence and absence of expected compounds. AE-MS also demonstrated its utility in relative quantifications of the same compound in serial dilution plates, or substrate in chemical synthesis. To facilitate the processing of a large amount of data generated by AE-MS, we have developed a data processing platform using commercially available tools. The platform demonstrated fast and straightforward data extraction, reviewing, and reporting, thus eliminating the need for the development of custom data processing tools. The overall AE-MS workflow has effectively eliminated the analytical bottleneck in the high-throughput compound QC work stream.

Enabling direct and definitive free fraction determination for highly-bound compounds in protein binding assay.

Protein binding determination for highly-bound compounds using equilibrium dialysis remains a challenge in drug discovery. The reasons are mainly three-fold; 1. due to their slow diffusion rate, highly-bound compounds require a much longer incubation time to reach dialysis equilibrium than typically needed; 2. highly-bound compounds are often hydrophobic and prone to non-specific binding in dialysis; 3. free drug concentration in the post incubation dialysate is too low for reliable analytical quantification. Modified equilibrium dialysis approaches include using diluted plasma for dialysis, or pre-saturating the non-specific binding sites in the dialysis device with compounds of interest prior to dialysis. In this study, we developed a customized equilibrium dialysis assay with an extended incubation time of 24 h, followed by microflow (µF) LC-MS/MS for bioanalysis, for direct and definitive free fraction determination of highly protein-bound compounds. The extended incubation time ensured the dialysis to reach equilibrium and saturating the non-specific binding sites, while µFLC-MS/MS provided far better sensitivity than the conventional LC-MS/MS typically used for post incubation bioanalysis. For a group of commercially available, highly protein-bound compounds, the free fraction data generated by the developed assay correlated very well with the literature values generated with diluted plasma method or pre-saturation method. This novel assay approach has been successfully used to generate protein binding results for highly-bound compounds to support ongoing drug discovery research.

Ultrahigh-Throughput and Chromatography-Free Bioanalysis of Polar Analytes with Acoustic Ejection Mass Spectrometry.

Bioanalysis of polar analytes using liquid chromatography-tandem mass spectrometry (LC-MS/MS) remains a significant challenge because of their poor chromatographic retention on the commonly used reversed-phase LC columns and the resulting severe ionization suppression from coeluting matrix components. Here we present a novel approach to perform ultrahigh-throughput and chromatography-free bioanalysis of polar compounds using a prototype acoustic ejection mass spectrometer (AEMS) platform. Previously developed for direct analysis of solid or liquid samples by MS, the open port interface (OPI) has recently been modified and coupled to an acoustic nanoliter dispenser to enable high-speed direct MS analysis from 384-well plates with a reported speed as fast as 0.5 s/sample. Ionization suppression was reduced due to the >1000 fold dilution of the original sample by the carrier solvent in the AE-OPI-MS operation. Taking full advantage of the chromatography-free and suppression-reducing features of this prototype instrument, we successfully demonstrated the ultrahigh-throughput bioanalysis of metformin, a small polar substrate commonly used in high-throughput in vitro transporter inhibition assays in the early ADME profiling space in drug discovery. The AEMS platform achieved a speed of 2.2 s/sample using only 10 nL of sample volume. Similar bioanalytical and biological results from actual assay samples were obtained by AEMS when compared to those obtained by the fastest LC-MS/MS method previously reported, along with a 15-fold speed advantage and ∼500-fold less sample consumption to enable future assay miniaturization. The general applicability of this novel approach to bioanalysis of several classes of polar analytes including ethambutol, isoniazid, ephedrine, and gemcitabine in biological matrices was further demonstrated.

Current status and future directions of high-throughput ADME screening in drug discovery.

During the last decade high-throughput in vitro absorption, distribution, metabolism and excretion (HT-ADME) screening has become an essential part of any drug discovery effort of synthetic molecules. The conduct of HT-ADME screening has been "industrialized" due to the extensive development of software and automation tools in cell culture, assay incubation, sample analysis and data analysis. The HT-ADME assay portfolio continues to expand in emerging areas such as drug-transporter interactions, early soft spot identification, and ADME screening of peptide drug candidates. Additionally, thanks to the very large and high-quality HT-ADME data sets available in many biopharma companies, in silico prediction of ADME properties using machine learning has also gained much momentum in recent years. In this review, we discuss the current state-of-the-art practices in HT-ADME screening including assay portfolio, assay automation, sample analysis, data processing, and prediction model building. In addition, we also offer perspectives in future development of this exciting field.