Biology subjects

Choi, M.

Publications and source records attributed to Choi, M..

At least 37 records · Page 2Linked to original sources

bioRxiv2025

Precise MRI-Histology Coregistration of Paraffin-Embedded Tissue with Blockface Imaging

Magnetic resonance imaging (MRI) provides 3D spatial information on tissue, yet it lacks at the molecular level. In contrast, histology provides cellular and molecular information, but it lacks the 3D spatial context and direct in vivo translation. Coregistering the two is key for the 3D-embedding of histological details, validating pathological MRI findings, and finding quantitative imaging biomarkers of neurodegenerative diseases. However, coregistration is challenging due to non-linear distortions of the tissue from histological processing and sectioning leading to microscopic and macroscopic nonlinear 3D deformations between specimen MRI and stained histology sections. To address this, we developed a novel pipeline, named Brewsters Blockface Quantification (BBQ), integrating robust optical approaches with innovative 2D and 3D registration algorithms to achieve precise volumetric alignment of specimen MRI data with histological images. On a variety of brain tissue specimens from distinct anatomical regions and across multiple species, our methodology generated blockface volumes with minimal distortion and artifacts. Using these blockface volumes as an intermediary, we achieve a precise alignment between MRI and histology slides, yielding registration results with an overlapping Dice score of [~]90% for whole tissue alignment between MRI and blockface volumes, and >95% for 2D MRI-histology registration. This correlative MRI-histology pipeline with robust 2D and 3D coregistration methods promises to enhance our understanding of neurodegenerative diseases and aid the development of MRI-based disease biomarkers.

bioengineering↗

bioRxiv2025

CoVET: Cryo-Electron Tomography guided by optical electrophysiology

Neurons exhibit varying electrophysiological properties due to dynamic changes in spatiotemporal molecular networks. In situ cryo-electron tomography (cryo-ET) provides unique advantages for high-resolution visualization of macromolecular complexes within their cellular context. Although correlation with fluorescent labeling allows cryo-ET to target specific cellular regions, it does not adequately reflect the electrophysiological properties of heterogeneous neurons. To bridge high-resolution molecular imaging with electrophysiological properties of individual neurons, we developed a Correlative-Voltage Imaging and cryo-ET (CoVET) technique. The nondestructive nature of voltage imaging is compatible with cryo-ET, enabling a direct correlation between neuronal electrophysiology and molecular structures. Neurons were clustered based on their electrophysiological properties, allowing for single-cell-guided structural analysis using cryo-ET. We analyzed the translational landscapes of individual neurons and found distinct translational profiles and contextual information among ribosomes from different electrophysiological clusters. Our results highlight the importance of the correlation between the electrophysiological properties and molecular structures.

biophysics↗

bioRxiv2025

Cell-Type-Specific Surfaceome Profiling of 100-500 Isolated Cells using a Droplet-Based Magnetic Affinity Purification System

Cell surface proteins (CSPs) represent an important source of biomarkers and therapeutic targets. However, due to the inherent sensitivity limitations of existing technologies, tissue and cell-type-specific surfaceomes remain poorly characterized, especially in the context of human diseases. Herein, we develop nanoMAPS (nanoscale Magnetic Affinity Purification System), a miniaturized proteomic sample preparation method for surfaceome profiling of as few as 100-500 cells (1000x to 100,000x lower than existing technologies). We demonstrate that the miniaturization of magnetic bead-based affinity purification inside a single droplet can efficiently improve the recovery of surface proteins and reduce non-specific absorption of intracellular proteins. By applying nanoMAPS to human immune cells isolated from PBMCs, we demonstrate robust identification of both well-known cell-type-specific surface markers and candidate proteins. We establish nanoMAPS as a promising platform to expand surface proteomics from cultured cells to primary cells isolated from patients or mouse models.

systems biology↗

bioRxiv2025

Combinatorial effects of Zingiber officinale and Citrus limon juices: Hypolipidemic and antioxidant insights from in vivo, in vitro, and in silico investigations

The combined use of Zingiber officinale (ginger) and Citrus limon (lemon) has long been valued in traditional medicine for their significant therapeutic properties, particularly in supporting cardiovascular health. However, their synergistic effects on hyperlipidemia and oxidative stress, both critical risk factors for cardiovascular diseases, remain underexplored. This study employed a multidisciplinary approach including in vivo (mice model with triton WR-1339-induced hyperlipidemia), in vitro (antioxidant assays such as DPPH and ABTS), and in silico (molecular docking targeting HMG-CoA reductase) to evaluate the hypolipidemic and antioxidant potential of ginger and lemon juices. Phytochemical analysis identified high concentrations of bioactive compounds, with ginger juice containing 6-gingerol (15.54% peak area) and lemon juice featuring hesperidin (13.85% peak area) and rutin (5.57% peak area). These compounds were instrumental in the observed pharmacological effects. The combined formulation significantly improved lipid profiles, reducing total cholesterol by 54.3%, triglycerides by 49.8%, and LDL by 58.1% while increasing HDL by 47.6% compared to hyperlipidemic controls (p < 0.05). Antioxidant assays revealed high free radical scavenging activity, with IC50 values of 30.92 {+/-} 2.00 {micro}g/mL for ABTS and 44.94 {+/-} 1.02 {micro}g/mL for DPPH in the combined formulation. Molecular docking confirmed strong binding affinities, with hesperidin and 6-gingerol showing binding energies of -10.4 kcal/mol and -9.6 kcal/mol, respectively, against HMG-CoA reductase. In conclusion, combining Zingiber officinale and Citrus limon juices demonstrates robust hypolipidemic and antioxidant effects, significantly outperforming the individual juices. These results support their potential as natural therapeutic agents for managing hyperlipidemia and oxidative stress. Further clinical studies are recommended to validate these promising findings and explore their application in nutraceutical development.

pharmacology and toxicology↗

bioRxiv2025

A cytoskeletal scaffold promotes motile cilia assembly by regulating transition-zone integrity

Motile cilia are eukaryotic organelles with essential chemo- and mechano-sensing functions across evolution, from single cell organisms to humans. Motile cilia of the mammalian nervous, respiratory and reproductive systems are characterized by unique motility proteins to generate fluid flow essential for transporting metabolites and removing mucus. The molecular mechanism of motile cilia biogenesis remains unknown. Here, we use mouse genetics, single-molecule motility assays, proteomics, high-resolution imaging, and in situ cryo-tomography to identify mammalian KIF27, a motor protein of the Kinesin-4 family and homologue of the Hedgehog pathway regulator COS2/KIF7, as a key regulator of motile cilia assembly. We show that KIF27 promotes the integrity of the transition zone, a diffusion barrier situated at the cilium base. Loss of KIF27 causes specific and profound defects in axonemal structure and disrupts cilia beating, which collectively lead to organismal phenotypes that recapitulate primary ciliary dyskinesia. We show that the motile properties of KIF27 are dispensable for its function in motile cilia biogenesis. Instead, KIF27 acts as a microtubule scaffold to regulate the transition zone architecture and enable correct ciliary incorporation of motility-generating proteins. Given that KIF27 homologues exist in different evolutionarily lineages, we propose that the ancestral activities of KIF27/KIF7 kinesins were to form a microtubule-associated scaffold for protein-protein interactions pertinent to cilia formation and signaling. The transition-zone associated KIF27 activities may represent a general building principle for motile cilia assembly in diverse species and cell types.

developmental biology↗

bioRxiv2024

Integrated analysis of COVID-19 multi-omics data for eQTLs reveals genetic mechanisms underlying disease severity

The global pandemic caused by the SARS-CoV-2 virus provided an unprecedented opportunity to investigate genetic factors influencing the disease severity of the viral infection. Despite a plethora of recent research on both SARS-CoV-2 and COVID-19, few have taken a systems biology approach to address individual-level variation, especially based on non-European populations. Accordingly, we analyzed multi-omics data generated at three timepoints from 193 Korean COVID-19 patients with mild or severe symptoms, composed of whole genome sequencing, blood-based single-cell RNA-sequencing (2.15M cells), 195 cytokine profiles, and human leukocyte antigen (HLA) allele data. We identified expression quantitative trait loci (eQTLs), disease severity interacting eQTLs (n = 388), and disease progression interacting eQTLs (n = 945) for various cell types. We elucidated a complex regulatory mechanism involving HLA genes and their targets, and identified genetic determinants of cytokine levels. Finally, we show how regulation of ieQTLs is established by upstream transcription factors (TFs), illustrating complex regulation of the IGFBP7 ieQTL by a combined action of two TFs, which is potentially important in conferring differential severity. This study illuminates an efficient molecular interrogation framework that can be applied toward understanding infectious disease progression in individuals of different genotypes.

genetics↗

bioRxiv2024

TEAD-targeting small molecules induce a cofactor switch to regulate the Hippo pathway

TEAD proteins are the main transcriptional effectors of the Hippo signaling pathway and a pharmacological target in oncology. Most TEAD-targeting small molecules act by disrupting interaction with the oncogenic transcriptional activators YAP and TAZ. Here, we describe an alternative mechanism for TEAD lipid pocket binding molecules. We report that select sulfonamide-containing compounds promote TEAD interaction with the transcriptional repressor VGLL4 to induce a small molecule-mediated cofactor switch from YAP to VGLL4. Chemically induced VGLL4-TEAD complexes counteract YAP activity at chromatin to repress pro-growth gene networks, including genes involved in cellular proliferation and mechanosignaling. VGLL4 is required for an anti-proliferative response to these select compounds, and genetic deletion of VGLL4 causes resistance to these molecules in vitro and in vivo. Our data reveal a category of molecules that facilitate the repressive VGLL4-TEAD interaction and open up new understandings for curbing the oncogenic activity of Hippo pathway deregulation.

cancer biology↗

bioRxiv2024

Characterization of Newly Discovered Polyester Polyurethane-degrading Methylobacterium Aquaticum Strain A1

In this study, we present Methylobacterium aquaticum A1, a novel strain capable of degrading polyester polyurethane (PE-PUR). The attachment of M. aquaticum A1 to PE-PUR and its degradation capabilities were verified using Scanning Electron Microscopy (SEM) and Fourier-Transform Infrared Spectroscopy (FT-IR). Analysis of the reference genome of M. aquaticum revealed genes encoding enzymes with potential PE-PUR degrading activity, including esterases, lipase, proteases and amidase such as tesA, pgpB, aes, aprE, lon, degQ, and gatA. An esterase activity assay using p-nitrophenyl acetate (p-NPA) showed increased ester bond-cleaving activity when M. aquaticum A1 was exposed to polyurethane diol (PU-diol), suggesting inducible enzymatic activity involved in PE-PUR degradation. These findings highlight the potential of M. aquaticum A1 as a promising biocatalyst for PE-PUR degradation. IMPORTANCEMicrobial biodegradation is increasingly recognized as a sustainable approach to addressing microplastic pollution. This study introduces M. aquaticum A1, a newly isolated strain capable of adhering to and degrading polyester polyurethane (PE-PUR), one of the most widely utilized plastics. To our knowledge, this is the first report of PE-PUR degradation by a member of the Methylobacterium genus. This paper provides a detailed characterization of M. aquaticum A1 and identifies several enzyme candidates--tesA, pgpB, aes, aprE, lon, degQ, and gatA--as potentially involved in the degradation process. Given that Methylobacterium species are known to be ecologically beneficial and inhabit diverse environments, the capacity of M. aquaticum A1 to degrade PE-PUR presents a promising strategy for mitigating microplastic pollution across a range of ecosystems.

microbiology↗

bioRxiv2024

Identification of genes conferring individual-level variation responsible for metabolic dysfunction-associated steatohepatitis using single-cell eQTL analysis

Metabolic dysfunction-associated steatotic liver disease (MASLD) is increasingly recognized for its medical and socioeconomic impacts, driven by diverse genetic and environmental factors. To address the urgent need for individually tailored therapies, we performed single-cell expression quantitative trait loci (sc-eQTL) analysis on liver biopsies from 25 MASLD patients and 23 controls. This approach identified over 3,500 sc-eQTLs across major liver cell types and cell state-interacting eQTLs (ieQTLs) with significant enrichment for disease heritability (for MASLD trait, ieQTL enrichment odds ratio = 10.27). We integrated transcription factors (TFs) as upstream regulators of ieQTLs, revealing 601 functional units ("quartets") composed of TFs, cell states, ieSNPs, and ieGenes. From these results, we pinpoint the loss of an eQTL in EFHD1 during hepatocyte maladaptation associated with genotype-specific regulation by FOXO1, further contributing to the risk of MASLD. Our approach underscores the role of eQTL analysis in capturing crucial genetic variations that influence gene expression and clinical outcomes in complex diseases.

genetics↗

bioRxiv2024

Systemic CD8+ T cell effector signature predicts prognosis of lung cancer immunotherapy

BackgroundWhile immune checkpoint inhibitors (ICIs) are adopted as standard therapy in non-small cell lung cancer (NSCLC) patients, factors that influence variable prognosis still remain elusive. Therefore, a deeper understanding is needed of how germline variants regulate the transcriptomes of circulating immune cells in metastasis, and ultimately influence immunotherapy outcomes. MethodsWe collected peripheral blood mononuclear cells (PBMCs) from 73 ICI-treated NSCLC patients, conducted single-cell RNA sequencing, and called germline variants via SNP microarray. Determination of expression quantitative trait loci (eQTL) allows elucidating genetic interactions between germline variants and gene expression. Utilizing aggregation-based eQTL mapping and network analysis across eight blood cell types, we sought cell-type-specific and ICI-prognosis-dependent gene regulatory signatures. ResultsOur sc-eQTL analysis identified 3,616 blood- and 702 lung-cancer-specific eGenes across eight major clusters and treatment conditions, highlighting involvement of immune-related pathways. Network analysis revealed TBX21-EOMES regulons activity in CD8+ T cells and the enrichment of eQTLs in higher-centrality genes as predictive factors of ICI response. ConclusionsOur findings suggest that in the circulating immune cells of NSCLC patients, transcriptomic regulation differs in a cell type- and treatment-specific manner. They further highlight the role of eQTL loci as broad controllers of ICI-prognosis-predicting gene networks. The predictive networks and identification of eQTL contributions can lead to deeper understanding and personalized ICI therapy response prediction based on germline variants.

genomics↗

bioRxiv2024

Magnetic Resonance Imaging of Gastric Motility in Conscious Rats

IntroductionGastrointestinal (GI) magnetic resonance imaging (MRI) can simultaneously capture gastric peristalsis, emptying, and intestinal filling and transit. Performing GI MRI with animals requires anesthesia, which complicates physiology and confounds interpretation and translation from animals to humans. This study aims to enable MRI in conscious rats, and for the first time, characterize GI motor functions in awake versus anesthetized conditions. MethodsWe acclimated rats to remain awake, still, and minimally stressed during MRI. We scanned 14 Sprague-Dawley rats in both awake and anesthetized conditions after voluntarily consuming a contrast-enhanced test meal. ResultsAwake rats remained physiologically stable during MRI, showed gastric emptying of 23.7{+/-}1.4% after 48 minutes, and exhibited strong peristaltic contractions propagating through the antrum with a velocity of 0.72{+/-}0.04 mm/s, a relative amplitude of 40.7{+/-}2.3%, and a frequency of 5.1{+/-}0.1 cycles per minute. In the anesthetized condition, gastric emptying was about half of that in the awake condition, likely due to the effect of anesthesia in halving the amplitudes of peristaltic contractions rather than their frequency (not significantly changed) or velocity. In awake rats, the intestine filled more quickly and propulsive contractions were more occlusive. ConclusionWe demonstrated the effective acquisition and analysis of GI MRI in awake rats. Awake rats show faster gastric emptying, stronger gastric contraction with a faster propagation speed, and more effective intestinal filling and transit, compared to anesthetized rats. Our protocol is expected to benefit future preclinical studies of GI physiology and pathophysiology.

bioengineering↗

bioRxiv2024

Inhibiting EZH2 complements steroid effects in Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is a devastating X-linked disorder caused by mutations in the dystrophin gene. Despite recent advances in understanding the disease etiology and applying emerging treatment methodologies, glucocorticoid derivatives remain the only general therapeutic option that can slow disease development. However, the precise molecular mechanism of glucocorticoid action remains unclear, and there is still need for additional remedies to complement the treatment. Here, using single-nucleus RNA-sequencing and spatial transcriptome analyses of human and mouse muscles, we investigated pathogenic features in DMD patients and palliative effects of glucocorticoids. Our approach further illuminated the importance of proliferating satellite cells, and revealed increased activity of a signal transduction pathway involving EZH2 in the patient cells. Subsequent administration of EZH2 inhibitors to Dmd mutant mice resulted in improved muscle phenotype through maintaining the immune-suppressing effect but overriding the muscle weakness and fibrogenic effects exerted by glucocorticoids. Our analysis reveals pathogenic mechanisms that can be readily targeted by extant therapeutic options for DMD. TeaserA survey of DMD tissues in human and mouse suggests EZH2 as a critical factor in DMD satellite cells; its inhibition resulted in better prognosis.

genetics↗

bioRxiv2024

The diversity of SNCA transcripts in neurons, and its impact on antisense oligonucleotide therapeutics

The role of the SNCA gene locus in driving Parkinsons disease (PD) through rare and common genetic variation is well-recognized, but the transcriptional diversity of SNCA in vulnerable cell types remains unclear. We performed SNCA long-read RNA sequencing in human dopaminergic neurons and show that annotated SNCA transcripts account for only 5% of expression. Rather, the majority of expression (75%) at the SNCA locus originates from transcripts with alternative 5 and 3 untranslated regions. Importantly, 10% originates from transcripts encoding open reading frames not previously annotated, which are translated and detectable in human postmortem brain. Defining the 3 untranslated regions enabled the rational design of antisense oligonucleotides targeting the majority of SNCA transcripts, leading to the effective reversal of PD pathology, including protein aggregation, mitochondrial dysfunction, and toxicity. Resolving the complexity of the SNCA transcriptional landscape impacts RNA therapies and highlights differences in protein isoforms and their contribution to disease.

neuroscience↗

bioRxiv2024

Selective enrichment of Methylococcaceae versus Methylocystaceae methanotrophs via control of methane feeding scheme

Methanotrophs are crucial in keeping environmental CH4 emissions in check. However, how different groups of methanotrophs contribute to this important role in different environmental settings remain ambiguous. Here, in a simplified laboratory setting of well-mixed batch reactors fed continuous flow of CH4-containing gas, methanotrophic microbiomes were enriched from paddy soils under six different incubation conditions prepared as combinations of two different CH4 mixing ratios (0.5% and 10% v/v) and three supplemented Cu2+ concentrations (0, 2, and 10 M). Monitoring of the temporal community shifts in the reactor microbiomes observed domination of Methylocystis spp. in all three reactors fed 0.5% v/v, as further supported by the analyses of pmoCAB genes in the shotgun metagenomes of the single-point samples from the same reactors. Copper deficiency did not select for mmoXYZ-possessing methanotrophs. Instead, a cluster of mbn genes with an abundance accounting for approximately 5% of Methylocystis population was identified, suggesting a comparative ecological importance of methanobactin in Cu-deficient methanotrophy over soluble methane monooxygenases. These findings highlight the importance of Methylocystis spp. in mitigating emissions from CH4 hotspots, e.g., landfills and rice paddies, and suggest the feasibility of directed enrichment/isolation of Methylocystis spp. for utilization in, for example, methanobactin and polyhydroxybutyrate production. SynopsisThis study reports enrichment of a complex soil microbiota with 0.5% methane resulting in dominance of a specific group of methane-consuming bacteria Methylocystis, highlighting their ecological significance as CH4 sink.

microbiology↗

bioRxiv2024

Gustatory-neuron-supplied R-spondin-2 is required for taste bud replenishment

Taste buds undergo continuous cell turnover throughout life, and taste cell replenishment relies strictly on innervation, a phenomenon first described almost 150 years ago. Recently, we provided evidence that R-spondin 2 (Rspo2) may be the long-sought gustatory neuron-supplied factor that regulates taste stem cell activity, via its interaction with taste stem/progenitor cell-expressed receptor Rnf43/Znrf3. Yet, whether gustatory-neuron-supplied Rspo2 is strictly required for taste tissue maintenance has not been resolved. Here, we set out to determine the necessity of gustatory-neuron-supplied Rspo2 in taste tissue homeostasis using genetic approaches. We used a mouse line that harbors the neomycin-resistance gene (NeoR) in one of the intron regions of the Rspo2 gene, which results in reduced expression of Rspo2. The number of taste buds is significantly reduced in these mice, compared to wild-type mice, in both anterior and posterior tongue. This phenotypic change was completely reversed by removing NeoR from the Rspo2 gene, thus making it normal. We also combined adeno-associated virus (AAV)-based delivery of Cre recombinase with a mouse line amenable to Cre-based ablation of the Rspo2 exons encoding the receptor-binding domains. Such deletion of Rspo2 in the nodose-petrosal-jugular ganglion complex led to nearly complete loss of taste buds in the circumvallate papilla. Thus, we demonstrate that Rspo2 is the long-sought gustatory-neuron-supplied factor that acts on taste stem cells to maintain taste tissue homeostasis. SignificanceWe have known for 150 years that innervation is required to induce and maintain cell replacement in taste buds. Until recently, the identity of the inducing factor produced by neurons was unknown. We have shown that R-spondin alone is sufficient to substitute for neuronal input to induce taste bud regeneration. Using a genetic loss-of-function approach, we now demonstrate that gustatory-neuron-expressed Rspo2 is required to maintain taste tissue homeostasis. Altogether, our work reveals that Rspo2 is the long-sought neuron-supplied factor that regulates the activity of taste stem/progenitor cells.

neuroscience↗

bioRxiv2024

Increased inflammatory signature in myeloid cells of non-small cell lung cancer patients with high clonal hematopoiesis burden

Clonal hematopoiesis of indeterminate potential (CHIP) allows estimation of clonal dynamics and documentation of somatic mutations in the hematopoietic system. Recent studies utilizing large cohorts of the general population and patients have revealed significant associations of CHIP burden with age and disease status, including in cancer and chronic diseases. An increasing number of cancer patients are treated with immune checkpoint inhibitors (ICI), but the association of ICI response in non-small cell lung cancer (NSCLC) patients with CHIP burden remains to be determined. We collected blood samples from 100 metastatic NSCLC patients before and after ICI for high-depth sequencing of the CHIP panel and 63 samples for blood single-cell RNA sequencing(scRNA-seq). Whole exome sequencing (WES) was performed in an independent replication cohort of 180 patients. The impact of CHIP status on the immunotherapy response was not significant. However, metastatic lung cancer patients showed higher CHIP prevalence (44/100 for patients vs 5/42 for controls; P = 0.01). In addition, lung squamous cell carcinoma patients showed increased burden of larger clones compared to lung adenocarcinoma patients (8/43 for LUSC vs 2/50 for LUAD; P = 0.04). Furthermore, single cell RNA-seq analysis of the matched patients showed significant enrichment of inflammatory pathways mediated by NF-B in myeloid clusters of the severe CHIP group. Our findings suggest minimal involvement of CHIP mutation and clonal dynamics during immunotherapy but a possible role of CHIP as an indicator of immunologic response in NSCLC patients.

genomics↗

bioRxiv2023

A nonenzymatic dependency on inositol-requiring enzyme 1 controls cancer cell cycle progression and tumor growth

Endoplasmic-reticulum resident inositol-requiring enzyme 1 (IRE1) supports protein homeostasis via a cytoplasmic kinase-RNase module. Known cancer dependency on IRE1 entails its enzymatic activation of the transcription factor XBP1s and of RNA decay. We discovered that some cancer cells require IRE1 but not its enzymatic activity. IRE1 knockdown, but not enzymatic inhibition or XBP1 disruption, increased DNA damage and chromosome instability while engaging the TP53 pathway and cyclin-dependent kinase inhibitors and attenuating cell cycle progression. IRE1 depletion downregulated factors involved in chromosome replication and segregation and in chromatin remodeling. Immunoelectron microscopy indicated that endogenous IRE1 can localize to the nuclear envelope. Thus, cancer cells can require IRE1 either enzymatically or nonenzymatically, with significant implications for IRE1s biological role and therapeutic targeting.

cancer biology↗

bioRxiv2023

Increasing the Throughput and Reproducibility of Activity-Based Proteome Profiling Studies with Hyperplexing and Intelligent Data Acquisition

Intelligent data acquisition (IDA) strategies, such as real-time database search (RTS), have improved the depth of proteome coverage for experiments that utilize isobaric labels and gas phase purification techniques (i.e., SPS-MS3). While most applications of IDA have been focused on the analysis of protein abundance, these approaches have recently been applied to activity-based proteome profiling (ABPP) studies aimed at characterization of protein site engagement by small molecules. In this work, we extend IDA capabilities offered by vendor software through a program called InSeqAPI. First, we demonstrate robust performance of InSeqAPI in the analysis of biotinylated cysteine peptides from ABPP experiments. Then, we describe PairQuant, a method within InSeqAPI designed for the hyperplexing approach that utilizes protein-level isotopic labeling and peptide-level TMT labeling. PairQuant allows for TMT analysis of 36 conditions in a single sample and achieves [~]98% coverage of both peptide pair partners in a hyperplexed experiment as well as a 40% improvement in the number of quantified cysteine sites compared to non-RTS acquisition. We applied this method in ABPP study of ligandable cysteine sites in the nucleus leading to an identification of additional druggable sites on protein-and DNA-interaction domains of transcription regulators and on nuclear ubiquitin ligases.

biochemistry↗