Project description:A collection of digital automated proteomic sample preparation protocols detail three optimized step-by-step methods to: (A) lyse Gram-negative bacteria and fungal cells; (B) quantify the amount of protein extracted; and (C) normalize the amount of protein and set up tryptic digestion. These protocols have been developed to facilitate rapid, low variance sample preparation of hundreds of samples, be easily implemented on widely-available Beckman-Coulter Biomek automated liquid handlers, and allow flexibility for future protocol development
Project description:Sample preparation is a crucial step in bottom-up proteomics. Analytical performances of bottom-up proteomics can be improved by the miniaturization of sample preparation steps. Many microfluidic devices are proposed in the field of proteomics. But many of them are not capable of handling complex sample and do not integrate the processing and digestion steps. We propose a ChipFilter Proteolysis (CFP) microfluidic device derived from the Filter Aided Sample Preparation FASP method for the miniaturization of protein processing and digestion steps in bottom-up proteomics. The microchip has two reaction chambers of 0.6 µL volume separated by a protein filtration membrane in regenerated cellulose. Cell lysis, protein concentration and rapid chemical and enzymatic treatment can be performed in our microfluidic device. Complex proteomic samples like yeast protein extract have already been analyzed with our microchip. Compared to the traditional FASP method, our microfluidic device offers a better proteome coverage with ten times less starting material and eight times quicker protocol.
Project description:Sample preparation is a crucial step in bottom-up proteomics. Analytical performances of bottom-up proteomics can be improved by the miniaturization of sample preparation steps. Many microfluidic devices are proposed in the field of proteomics. But many of them are not capable of handling complex sample and do not integrate the processing and digestion steps. We propose a ChipFilter Proteolysis (CFP) microfluidic device derived from the Filter Aided Sample Preparation FASP method for the miniaturization of protein processing and digestion steps in bottom-up proteomics. The microchip has two reaction chambers of 0.6 µL volume separated by a protein filtration membrane in regenerated cellulose. Cell lysis, protein concentration and rapid chemical and enzymatic treatment can be performed in our microfluidic device. Complex proteomic samples like yeast protein extract have already been analyzed with our microchip. Compared to the traditional FASP method, our microfluidic device offers a better proteome coverage with ten times less starting material and eight times quicker protocol.
Project description:Selecting a sample preparation strategy for mass spectrometry-based proteomics is critical to the success of quantitative workflows. Here we present a universal, solid-phase protein preparation (USP3) method which is rapid, robust and scalable, facilitating high-throughput protein sample preparation for bottom-up and top-down mass spectrometry (MS) analysis.
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.