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:We investigated the effects of different sample preparation factors on RNA-seq experiments, including RNA concentration, library storage time, and cryopreserved condition, by comparing their sequencing biases, gene expression profiles, and biological function using primary B cell and CD4+ cell blood samples in healthy subjects.
Project description:DNA replication occurs in a defined temporal order known as the replication-timing (RT) program. RT is regulated during development in discrete chromosomal units, coordinated with transcriptional activity and 3D genome organization. Here, we developed a sample preparation method to measure RT genome-wide that does not require fixation.