US HUPO 2021
Join us at this year’s US HUPO. We are very much present at this years conference, and even though we miss traveling the globe to meet fellow researchers, we have an exciting program lined up with talks from both Evosep and our users. Learn about all our activities below.
Parallel Session 01: Single Cell Proteomics
March 8, 2021 at 9:00am – 10:30am Pacific / 12:00pm – 1:30pm Eastern
#96 Robust, very low nanoflow LC-MS methods for ultra-sensitive single-cell and phosphoproteomics
Talk by Dorte Bekker-Jensen, senior scientist, evosep
Ultra-sensitive proteomics is a rapidly growing field where cutting-edge technologies are pushed to their limits for applications such as single-cell analyses. One of the main challenges to achieve this, is to maintain sensitivity and robustness throughout the entire workflow, where attention is especially needed during sample preparation and efficient loading of minute sample amounts to the LC-MS system. Here we describe how the Evotip, a disposable trap column, is an integral part for efficient concentration and loading of very low sample amounts. As the electrospray ionization efficiency is concentration dependent, we achieve the highest sensitivity at low flowrates. To take advantage of this, we developed a set of LC methods operating at gradient flows of 25, 50 and 100 nl/min on standard Evosep One hardware. This setup builds on a robust platform allowing for several thousand uninterrupted injections even at very low flow rates.
To verify the expected increase in sensitivity with the nanoflow methods, we performed dilution series of HeLa digests, analyzed the samples with the 30 min method operating at a flow rate of 100 nl/min and benchmarked its performance directly against the established 60 samples per day method, with a gradient flow of 1 ul/min. We found that at sub-ng of HeLa peptides, the nanoflow method increased up to ten-fold compared to the standard method, resulting in more than 50% increase in protein identifications. Interestingly, these proteins were also measured with significantly higher run-to-run reproducibility.
We also challenged our method by analyzing phosphopeptides enriched from just 1ug of peptide starting material. Again, we found a factor of 10x higher average intensity compared to the standard method. In conclusion, the combination of using Evotips and preformed gradients allow operation at extremely low flow rates with undiminished robustness, opening exciting application areas.
Tuesday Evosep Lunch seminar
Ultra sensitive and high throughput proteomics on the standardized Evosep One platform
March 9, 2021 at 10:00am – 11:00am ET
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
Spatial-proteomics reveal in-vivo phospho-signaling dynamics at subcellular resolution
Talk by Jesper Olsen, Group leader at Olsen Group, University of Copehagen
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.
Bruker: Seminar with Dr. Kirk Hansen and Chris Adams
March 9, 2021 at 2.00pm ET
Plasma proteomics during a pandemic with a focus on covid-19 and trauma patients
Talk by Dr. Kirk Hansen, Professor at University at Colorado
Over 100 million people around the world have tested positive for the beta coronavirus SARS-CoV-2 from late 2019 to February 1st, 2021. COVID-19 has been characterized by elevated mortality with approximately 2.24 million deaths, primarily due to thrombo-inflammatory complications that impair lung perfusion and systemic oxygenation in the most severe cases. Over the same time period we developed a plasma proteomics pipeline using the Evosep One and timsTOF Pro system to explore systemic alterations in numerous injury and disease states. Similarities between COVID-19 and trauma, the number one cause of death for individuals under 45 with approximately 5.8 million annual deaths worldwide, lead to the exploration of a therapeutic intervention that activates the fibrinolytic system.