Julia Robbins is a senior research associate at Talus Bio with five years of experience in mass spectrometry proteomics. Her research focuses on methods development for high-throughput proteomics sample preparation, and she has particular interest in bead-based automation. When she’s not orchestrating multiple robots and instruments in the lab, she’s a doting plant mom and dabbles in scientific illustration.
Protein localization within the subcellular environment influences their structure and function. One example is the transcription factor (TF) protein class, which commonly reside in the cytosol prior to being activated, typically by post-translational modification, and then translocating to the nucleus where they interact with DNA and other regulatory proteins to initiate transcription of genes. However, studying TFs is difficult because the process of isolating nuclei is a low-throughput, manual process, and existing bead-based approaches are not selective for nuclei.
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We developed a novel lectin-based magnetic bead approach to manipulate nuclei that is more selective and higher-throughput than other methods, using Skyline for glycan and proteomics analyses throughout the method development process. We used SUGA to prepare glycans from the nucleus and from the cytosol, and Skyline to determine which of those glycans was differentially localized to the nucleus. We prepared magnetic beads with lectins specific to those glycans, and used a targeted Skyline document with proteins from different cellular compartments to quantify the enrichment of nuclei using those lectin beads. Finally, we set up AutoQC and Panorama with the Skyline document for subcellular fractionation in order to monitor for nuclear enrichment across a 96-well plate based method using the magnetic beads.
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