Project description:The analysis of single cell proteomes has recently become a viable complement to transcript and genomics studies. Proteins are the main driver of cellular functionality and mRNA levels are often an unreliable proxy of such. Therefore, the global analysis of the proteome is essential to study cellular identities. Both multiplexed and label-free mass spectrometry-based approaches with single cell resolution have lately attributed surprising heterogeneity to believed homogenous cell populations. Even though specialized experimental designs and instrumentation have demonstrated remarkable advances, the efficient sample preparation of single cells still lacks behind. Here, we introduce the proteoCHIP, a universal option for single cell proteomics sample preparation at surprising sensitivity and throughput. The automated processing using a commercial system combining single cell isolation and picoliter dispensing, the cellenONE®, allows to reduce final sample volumes to low nanoliters submerged in a hexadecane layer simultaneously eliminating error prone manual sample handling and overcoming evaporation. With this specialized workflow we achieved around 1,000 protein groups per analytical run at remarkable reporter ion signal to noise while reducing or eliminating the carrier proteome. We identified close to 2,000 protein groups across 158 multiplexed single cells from two highly similar human cell types and clustered them based on their proteome. In-depth investigation of regulated proteins readily identified one of the main drivers for tumorigenicity in this cell type. Our workflow is compatible with all labeling reagents, can be easily adapted to custom workflows and is a viable option for label-free sample preparation. The specialized proteoCHIP design allows for the direct injection of label-free single cells via a standard autosampler resulting in the recovery of 30% more protein groups compared to samples transferred to PEG coated vials. We therefore are confident that our versatile, sensitive, and automated sample preparation workflow will be easily adoptable by non-specialized groups and will drive biological applications of single cell proteomics.
Project description:Recent advances in stem cell technology have led to the development of three-dimensional (3D) culture systems called organoids, which have fueled hopes to bring about the next generation of more physiologically relevant high throughput screens (HTS). However, the adaptation of established organoid protocols for HTS applications has so far been elusive. Here, we present a fully scalable, HTS-compatible workflow for the automated generation, maintenance, whole mount staining, clearing, and optical analysis of human neural organoids generated from neural precursor cells in a standard 96-well format. By combining organoid generation and analysis steps in an automated fashion, we can perform quantitative whole-organoid high content imaging with single cell resolution. The resulting organoids are highly homogeneous with regard to their morphology, size, global gene expression, cellular composition, and structure. Calcium imaging suggests organoid-wide synchronized functional coupling. The scalability of our approach has the potential to form the basis for 3D tissue-based screening in a variety of applications including drug development, toxicology studies, and disease modeling.
Project description:Bottom-up proteomics holds significant promise for clinical applications due to its high sensitivity and precision, but is limited by labor-intensive, low-throughput sample preparation meth-ods. Advanced automation is essential to enhance throughput, reproducibility, and accuracy and to allow standardization, thus making bottom-up proteomics amenable for large-scale studies. We developed a fully integrated, automated sample preparation platform that covers the entire process from biological sample input to mass spectrometry-ready peptide output and can be applied on a multitude of biological samples. With this end-to-end solution, we achieved high intra- and interplate reproducibility, as well as longitudinal consistency, result-ing in precise and reproducible workflows. We showed that our automated workflow surpass-es established manual and semi-automated workflows, while improving time efficiency. Finally, we demonstrated the suitability of our automated sample preparation platform for drug development by performing a high-content compound characterization for targeted pro-tein degradation . For this, we coupled application-specific workflows to perform high-content compound characterization by proteome profiling and confirm target degra-dation by precise and reliable protein quantification. Thus, the automated sample preparation platform facilitates rapid adaptation to emerging developments in proteomics sample prepara-tion, combining standardization, flexibility, and high-throughput capabilities to drive significant advancements in clinical assays and proteomics research.
Project description:Bottom-up proteomics holds significant promise for clinical applications due to its high sensitivity and precision, but is limited by labor-intensive, low-throughput sample preparation meth-ods. Advanced automation is essential to enhance throughput, reproducibility, and accuracy and to allow standardization, thus making bottom-up proteomics amenable for large-scale studies. We developed a fully integrated, automated sample preparation platform that covers the entire process from biological sample input to mass spectrometry-ready peptide output and can be applied on a multitude of biological samples. With this end-to-end solution, we achieved high intra- and interplate reproducibility, as well as longitudinal consistency, result-ing in precise and reproducible workflows. We showed that our automated workflow surpass-es established manual and semi-automated workflows, while improving time efficiency. Finally, we demonstrated the suitability of our automated sample preparation platform for drug development by performing a high-content compound characterization for targeted pro-tein degradation . For this, we coupled application-specific workflows to perform high-content compound characterization by proteome profiling and confirm target degra-dation by precise and reliable protein quantification. Thus, the automated sample preparation platform facilitates rapid adaptation to emerging developments in proteomics sample prepara-tion, combining standardization, flexibility, and high-throughput capabilities to drive significant advancements in clinical assays and proteomics research.
Project description:Bottom-up proteomics holds significant promise for clinical applications due to its high sensi-tivity and precision, but is limited by labor-intensive, low-throughput sample preparation meth-ods. Advanced automation is essential to enhance throughput, reproducibility, and accuracy and to allow standardization, thus making bottom-up proteomics amenable for large-scale studies. We developed a fully integrated, automated sample preparation platform that covers the entire process from biological sample input to mass spectrometry-ready peptide output and can be applied on a multitude of biological samples. With this end-to-end solution, we achieved high intra- and interplate reproducibility, as well as longitudinal consistency, result-ing in precise and reproducible workflows. We showed that our automated workflow surpass-es established manual and semi-automated workflows, while improving time efficiency. Finally, we demonstrated the suitability of our automated sample preparation platform for drug development by performing a high-content compound characterization for targeted pro-tein degradation . For this, we coupled application-specific workflows to perform high-content compound characterization by proteome profiling and confirm target degra-dation by precise and reliable protein quantification. Thus, the automated sample preparation platform facilitates rapid adaptation to emerging developments in proteomics sample prepara-tion, combining standardization, flexibility, and high-throughput capabilities to drive significant advancements in clinical assays and proteomics research.
Project description:Bottom-up proteomics holds significant promise for clinical applications due to its high sensitivity and precision, but is limited by labor-intensive, low-throughput sample preparation meth-ods. Advanced automation is essential to enhance throughput, reproducibility, and accuracy and to allow standardization, thus making bottom-up proteomics amenable for large-scale studies. We developed a fully integrated, automated sample preparation platform that covers the entire process from biological sample input to mass spectrometry-ready peptide output and can be applied on a multitude of biological samples. With this end-to-end solution, we achieved high intra- and interplate reproducibility, as well as longitudinal consistency, result-ing in precise and reproducible workflows. We showed that our automated workflow surpass-es established manual and semi-automated workflows, while improving time efficiency. Finally, we demonstrated the suitability of our automated sample preparation platform for drug development by performing a high-content compound characterization for targeted pro-tein degradation . For this, we coupled application-specific workflows to perform high-content compound characterization by proteome profiling and confirm target degra-dation by precise and reliable protein quantification. Thus, the automated sample preparation platform facilitates rapid adaptation to emerging developments in proteomics sample prepara-tion, combining standardization, flexibility, and high-throughput capabilities to drive significant advancements in clinical assays and proteomics research.
Project description:Liquid handling robots have been developed to automate various steps of the bottom-up proteomics workflow, however, protocols for the generation of isobarically labeled peptides remain limited. Existing methods often require costly specialty devices and are constrained by fixed workflows. To address this, we developed a cost-effective, flexible, automated sample preparation protocol for TMT-labeled peptides using the Biomek i5 liquid handler. Our approach leverages Single-Pot Solid-Phase-Enhanced Sample Preparation (SP3) with paramagnetic beads to streamline protein cleanup and digestion. The protocol also allows for adjustment of trypsin concentration and peptide-to-TMT ratio to increase throughput and reduce costs, respectively. We compared our automated and manual 18-plex TMT-Pro labeling workflows by monitoring select protein markers of the Unfolded Protein Response (UPR) in pharmacologically activatable, engineered cell lines. Overall, the automated protocol demonstrated equivalent performance in peptide and protein identifications, digestion and labeling efficiency, and an enhancement in the dynamic range of TMT quantifications. Compared to the manual method, the Biomek protocol significantly reduces hands-on time and minimizes sample handling errors. The 96-well format additionally allows for the number of TMT reactions to be scaled up quickly without a significant increase in user interaction. Our optimized automated workflow enhances throughput, reproducibility, and cost-effectiveness, making it a valuable tool for high-throughput proteomics studies.
Project description:Although tandem mass tag (TMT)-based isobaric labeling has become a powerful technique for multiplexed protein quantitation, it has not been easy to automate the workflow for widespread adoption. This is because preparation of TMT labeled peptide samples involves multiple steps ranging from protein extraction, denaturation, reduction and alkylation to tryptic digestion, desalting, labeling with TMT reagents and cleanup, all of which require a high level of proficiency. The variability resulting from multiple processing steps is inherently problematic especially with large-scale studies such as clinical studies that involve hundreds of samples where reproducibility is critical for quantitation. Here, we sought to compare the performance of a recently introduced platform, AccelerOme, for automated proteomics workflows for TMT-labeling experiments with manual processing of samples. Cell pellets were prepared and subjected to a 16-plex experiment using the automated platform and a conventional manual protocol. Single shot LC-MS/MS analysis revealed a higher number of proteins and peptides identified using the automated platform. Efficiencies of tryptic digestion, alkylation and TMT labeling were similar both in manual and automated process. In addition, comparison of quantitation accuracy and precision showed similar performance in automated workflow compared to manual sample preparation. Overall, we demonstrated that the automated platform performs at a level similar to manual process in TMT-based proteomics. We expect that the automated workflow will increasingly replace manual work and be applied to large-scale TMT-baed studies providing robust results and high sample throughput.