Automated Liquid Handling for
Assay Development and Optimization

Achieving high-resolution spatial tissue proteomes requires careful balancing and integration of optimized sample processing chromatography and MS acquisition Here we present an advanced cellenONE protocol for loss-reduced tissue processing and compare all Evosep ONE Whisper Zoom gradients and samples per day along with three common DIA acquisition schemes on a ...More |Related Solutions: Mantis^®

Achieving high-resolution spatial tissue proteomes requires careful balancing and integration of optimized sample processing, chromatography, and MS acquisition. Here, we present an advanced cellenONE protocol for loss-reduced tissue processing and compare all Evosep ONE Whisper Zoom gradients (20, 40, 80, and 120 samples per day), along with three common DIA acquisition schemes on a timsUltra AIP mass spectrometer. We found that tissue type was as important as gradient length and sample amount in determining proteome coverage. Moreover, the benefit of increased tissue sampling was gradient- and dynamic range-dependent. Analyzing mouse liver, a high dynamic range tissue, over tenfold more tissue sampling led to only ~30% gain in protein identification for short gradients (120 SPD and 80 SPD). However, even the lowest tested tissue amount (0.04 nL, 40,000 µm3) yielded 3,200 reproducibly quantified proteins for the 120 SPD method. Longer gradients (40 SPD and 20 SPD) instead significantly benefited from more tissue sampling, quantifying over 7,500 proteins from 0.5 nL of tonsil T-cell niches. Finally, we applied our workflow to a rare squamous cell carcinoma of the oral cavity, uncovering disease-associated pathways and region-specific protein level changes. Our study demonstrates that more than 100 high-quality spatial tissue proteomes can be prepared and acquired daily, laying a strong foundation for cohort-size spatial tissue proteomics in translational research. Less |Related Solutions: Mantis^®

Prophages dormant bacteriophage genomes integrated within the bacterial chromosome play pivotal roles in shaping microbial communities when awakened Our current understanding of prophage activation is largely shaped by a narrow set of traditional DNA-damaging inducers such as mitomycin C and ciprofloxacin which trigger the bacterial SOS response This study employed ...More |Related Solutions: Tempest^®

Prophages, dormant bacteriophage genomes integrated within the bacterial chromosome, play pivotal roles in shaping microbial communities when awakened. Our current understanding of prophage activation is largely shaped by a narrow set of traditional DNA-damaging inducers, such as mitomycin C and ciprofloxacin, which trigger the bacterial SOS response. This study employed high-throughput screening of 3,921 compounds to identify novel prophage inducers using model lambdoid prophage HK97. We identified multiple new inducers across diverse pharmacological classes, including dietary supplements and therapeutics. Despite the variety in compounds, all acted through SOS-dependent pathways. However, bleomycin, an antineoplastic antibiotic, demonstrated broad-spectrum and potent prophage induction exceeding standard inducers, with activity validated across multiple phage-host pairings. These findings expand the repertoire of prophage inducers into commonly ingested xenobiotics and introduce bleomycin as a powerful, cost-effective tool for prophage research.
Around 75% of bacteria carry within them dormant viruses (prophages), which can awaken when the bacterium is stressed, killing the bacterium. Historically, this has been done using DNA-damaging antibiotics, but increasingly, more such signals have been discovered. Here, through a high-throughput screen, we identify phage-waking activity in several commonly consumed compounds, such as the SSRI Prozac, as well as a new DNA-damaging agent that is much more effective in waking phages than the previous gold standard. Less |Related Solutions: Tempest^®

Plasma and cerebrospinal fluid are complementary sources of biomarkers for neurodegenerative diseases The wide dynamic range of protein abundances particularly in plasma hampers detection of low-abundance proteins Depletion of high-abundance proteins and efficient enzymatic digestion can improve proteome coverage but must be carefully optimized for reproducibility throughput and cost-efficiency for ...More |Related Solutions: Mantis^®

Plasma and cerebrospinal fluid are complementary sources of biomarkers for
neurodegenerative diseases. The wide dynamic range of protein abundances, particularly in
plasma, hampers detection of low-abundance proteins. Depletion of high-abundance proteins
and efficient enzymatic digestion can improve proteome coverage but must be carefully
optimized for reproducibility, throughput, and cost-efficiency for use in large-scale clinical
proteomic studies.
We developed a scalable sample preparation workflow for plasma and cerebrospinal fluid (CSF)
that integrates depletion of high-abundance proteins, optimized digestion using Lys-C and
trypsin, and compatibility with both label-free and tandem mass tag (TMTpro)-based
quantification. We systematically evaluated protein depletion and enzyme digestion conditions,
and the effect of deoxycholate on digestion, monitoring the number of detectable proteins and
the quantitation precision. Less |Related Solutions: Mantis^®

The Maternal-to-Zygotic transition MZT is a reprograming process encompassing zygotic genome activation ZGA and the clearance of maternally-provided mRNAs While some factors regulating MZT have been identified there are thousands of maternal RNAs whose function has not been ascribed yet Here we have performed a proof-of-principle CRISPR-RfxCas d maternal screening ...More |Related Solutions: Mantis^®

The Maternal-to-Zygotic transition (MZT) is a reprograming process encompassing zygotic genome activation (ZGA) and the clearance of maternally-provided mRNAs. While some factors regulating MZT have been identified, there are thousands of maternal RNAs whose function has not been ascribed yet. Here, we have performed a proof-of-principle CRISPR-RfxCas13d maternal screening targeting mRNAs encoding protein kinases and phosphatases in zebrafish and identified Bckdk as a novel post-translational regulator of MZT. Bckdk mRNA knockdown caused epiboly defects, ZGA deregulation, H3K27ac reduction and a partial impairment of miR-430 processing. Phospho-proteomic analysis revealed that Phf10/Baf45a, a chromatin remodeling factor, is less phosphorylated upon Bckdk depletion. Further, phf10 mRNA knockdown also altered ZGA and Phf10 constitutively phosphorylated rescued the developmental defects observed after bckdk mRNA depletion. Altogether, our results demonstrate the competence of CRISPR-RfxCas13d screenings to uncover new regulators of early vertebrate development and shed light on the post-translational control of MZT mediated by protein phosphorylation. Less |Related Solutions: Mantis^®

The increase in multidrug-resistant bacteria underscores an urgent need for additional antibiotics Here we integrate small-molecule high-throughput screening with a deep-learning-based virtual screening approach to uncover new antibacterial compounds We screen million small molecules against a sensitized Escherichia coli strain yielding thousands of hits We use these data to train ...More |Related Solutions: Tempest^®

The increase in multidrug-resistant bacteria underscores an urgent need for additional antibiotics. Here, we integrate small-molecule high-throughput screening with a deep-learning-based virtual screening approach to uncover new antibacterial compounds. We screen ~2 million small molecules against a sensitized Escherichia coli strain, yielding thousands of hits. We use these data to train a deep learning model, GNEprop, to predict antibacterial activity, retrospectively validating robustness with respect to out-of-distribution generalization and activity cliff prediction. Virtual screening of over 1.4 billion synthetically accessible compounds identifies potential candidates, of which 82 exhibit antibacterial activity on the same strain, illustrating a 90-fold improved hit rate over the high-throughput screening experiment used for training. Many newly identified compounds exhibit high dissimilarity to known antibiotics, potency beyond the training bacterial strain and selectivity. Biological characterization identifies specific, validated targets, indicating promising avenues for further exploration in antibiotic discovery. Less |Related Solutions: Tempest^®