US HUPO 2021: Evosep Seminar

ULTRA SENSITIVE AND HIGH THROUGHPUT PROTEOMICS ON THE STANDARDIZED EVOSEP ONE PLATFORM The recently introduced Whisper™ Flow Technology uses preformed gradients and disposable Evotip trap columns with the sample preloaded and immobilized to robustly and reproducibly handle verysmall sample amounts, down to actualsingle cells, at sub-100 nl/min flowrates. This maximizes sensitivity whilemaintaining the other advantages of the Evosep One, especially low time overheads, efficient acquisition windowuse, and low retention time variations. SPEAKERS SPATIAL-PROTEOMICS REVEAL IN-VIVO PHOSPHO-SIGNALING DYNAMICS AT SUBCELLULAR RESOLUTION TALK BY JESPER OLSEN, GROUP LEADER AT OLSEN GROUP, UNIVERSITY OF COPENHAGEN We present a streamlined and high-throughput spatial proteomics approach for analyzing subcellular phosphoproteome dynamics in a temporal manner. The method is based on sequential cell fractionation using different biochemical buffers to profile six distinct subcellular fractions with high reproducibility. Fast chromatographic separation using the Evosep One coupled to directDIA measuremens on an Orbitrap Exploris 480 mass spectrometer allows acquisition of complete data for both subcellular proteome and phosphoproteome in just 4 hours of MS time. Spatio-temporal analysis of the osmotic shock response and EGF signaling in cancer cells and in-vivo in mouse tissues reveals new interesting biology. LEVERAGING THE POWER OF WHISPER™ FOR HIGH-THROUGHPUT SINGLE CELL PROTEOMICS TALK BY ERWIN M. SCHOOF, ASSOCIATE PROFESSOR IN SINGLE-CELL PROTEOMICS IN DISEASE BIOLOGY / HEAD OF DTU PROTEOMICS CORE, DTU – TECHNICAL UNIVERSITY OF DENMARK We have taken a primary leukemia hierarchy, “OCI-AML8227”, and subjected it to our recently developed multiplexed single-cell proteomics (scMS) strategy to identify the protein landscapes of individual cells within this aberrant hierarchy. By using the latest state-of-the-art LC-MS instrumentation with intelligent data acquisition, combined with specialized Evosep Whisper™ gradients, one can, in an informed manner, strike a careful balance between unprecedented throughput and proteome depth of single cells. Furthermore, we developed a computational pipeline (SCeptre) that effectively normalizes the data, clusters the cells, integrates available FACS data and permits the extraction of cell-specific proteins. The approach presented here lays a solid foundation for implementing global scMS studies in labs across the world.