Introduction

Presented by Sameer Vasantgadkar,
Senior Manager, Omics Solutions, Covaris, Woburn, MA

Learn about specific, customizable workflows and kits for extraction, purification (with PAC protocol) and accelerated digestion of proteins from a variety of matrices. 

Leveraging Plate-Based AFA For Robust and Consistent High Throughput Proteomics 
Presented by Craig Braun,
Co-Founder, IQ Proteomics LLC, Cambridge, MA

Establishing robust and consistent plate-based sample processing workflows compatible with a wide range of sample types is crucial for enabling high-quality, high-throughput proteomics.  At IQ Proteomics, we leverage plate-based AFA technology enabled by the Covaris R230 in several components of our proteomics workflows to achieve this goal.  AFA lysis and homogenization of cell pellets and mammalian tissues proceeds simply and reproducibly while keeping volumes small and incorporating plate-based sample handling as early in the process as possible.  AFA also streamlines and improves downstream bead-based sample cleanup and digestion workflows (SP3). Incorporation of AFA into multiple points of the proteomics sample preparation workflow improves robustness, while increasing throughput.  Finally, R230 integration with robotic liquid handling during both lysis and bead-based protein cleanup/digestion enables start to finish automation of proteomics sample prep for high throughput proteomics applications. 

Proteomics Analysis of Limited Number of Cells by a Rapid and Efficient Workflow using Adaptive Focused Acoustic Technology
Presented by Anu Jain,
Research Fellow, Mayo Clinic, Rochester, MN

Characterization of cellular proteomes is key to understanding biological processes. With significant improvements in sensitivity and performance of mass spectrometers, it has become feasible to explore the cellular proteome when limited numbers of cells are available for analysis. However, this is still challenging, especially in terms of sample preparation strategies. Traditional multi-step sample processing strategies can result in sample loss, lower protein recovery and limited protein identification when starting with low cell numbers. In addition, longer processing times may limit the number of samples that can be analyzed. Our objective was to overcome some of these limitations by implementing a novel sample preparation strategy for proteomics analysis when low number of cells are available.

High-Throughput and Automated Cellular and Tissue Lysis using COVARIS Acoustic Technology for Proteomics 
Presented by Josselin Ortiz,
Biomedical Technician, Precision Biomarker Laboratories, Cedars-Sinai Medical Center, Beverly Hills, CA

Sample preparation is a key step in a proteomics workflow, particularly in clinical settings where biospecimens are limited. Furthermore, reproducibility is the key measure for locking down a developed workflow in translational research. A variety of proteomics sample preparation methods (ranging from laborious to automated) have been used to generate protein expression profiles for many types of cells and tissues. The main challenges in different methods include low reproducibility, proneness to error, and throughput issues. This study utilizes the Covaris LE220-plus instrument to assess the effective lysis of HEK293 cells and mice liver tissue with an increasing sample amount, followed by automated SP3 digestion and LC-MS/MS analysis. 

Introduction

Presented by Deb Bhattacharyya,
VP Business Development and Emerging Markets, Covaris, Woburn, MA

From the use of an advanced deparaffinization and protein extraction protocol to employing methods like PAC or S-Trap for purification to facilitating rapid digestion with Trypsin or Trypsin-Lys C, FFPE samples can now exhibit very high protein coverage. Find out how. 

High-Throughput, Streamlined Processing Workflow of Formalin-Fixed Paraffin-Embedded (FFPE) Tissue Yielding up to 10,000 Proteins per Sample 
Presented by Moe Haines,
Senior Research Associate II, Broad Proteomics, Broad Institute of MIT & Harvard, Cambridge, MA

Formalin-fixed paraffin-embedded (FFPE) tissue has long served as the standard for tissue preservation, representing an invaluable resource for clinical studies due to its association with patient outcomes and its link to molecular insights. Here we present a streamlined proteomic workflow aimed at overcoming current challenges associated with FFPE processing. The workflow employs the Covaris LE220-plus focused-ultrasonicator and Adaptive Focused Acoustics (AFA) technology for tissue lysis, plate-based S-Trap for proteolytic digestion, and automation for faster, more reproducible analyses. Samples are analyzed by data-independent acquisition (DIA) and are benchmarked against a TMT-based multiplexed workflow. This approach identifies up to ~10,000 unique proteins from single-shot injections using the Orbitrap Astral MS, with a turnaround of 4 days for a full 96-well plate. 

Adapting sample preparation techniques of Formalin-Fixed-Paraffin-Embedded (FFPE) tissue samples for high throughput proteomics: Challenges and opportunities 

Presented by Lynn Spruce,
Technical Director of the CHOP-PENN Proteomics Core Facility at the Children’s Hospital of Pennsylvania (CHOP) and University of Pennsylvania, Pennsylvania, PA

Crucial for gaining biological insights from proteomics data sets, there is a need to analyze ever larger numbers of biological replicates across patient populations to increase the statistical power. Instrumentation and analysis techniques have evolved to allow for proteomic analysis of large-scale experiments. Complete, reproducible extraction of proteins from tissue samples is a crucial first step for proteomic analysis. Availability of fresh/frozen tissues is often limited, and such material is not easily stored at scale. FFPE tissue samples collected over long periods of time are stored in bio banks worldwide and represent a deep reservoir of potential samples. No fully standardized protocol exists for protein extraction off FFPE tissues that would allow for more efficient, routine interrogation of large-scale sample cohorts. Our discussion will focus on our experiences of working with FFPE tissue samples at scale.

Proteomics Analysis of Limited Number of Cells by a Rapid and Efficient Workflow using Adaptive Focused Acoustic Technology
Presented by Anu Jain,
Mayo Clinic, Rochester, MN 

• TP 339
• Tuesday, June 4, 10:30-11:30, 12:30-2:30

Comprehensive Workflow for Extraction, Purification, and Accelerated Digestion of Proteins from Mammalian Cells
Presented by Deb Bhattacharyya,
Covaris, Woburn, MA

• WP 109
• Wednesday, June 5, 10:30-11:30, 12:30-2:30

Comprehensive Sample Preparation Workflow for Deparaffinizaton, Extraction, Purification, and Accelerated Digestion of Proteins from Formalin-Fixed Paraffin-Embedded Tissue
Presented by Sameer Vasantgadkar,
Covaris, Woburn, MA

• WP 185
• Wednesday, June 5, 10:30-11:30, 12:30-2:30

High-Throughput, Streamlined Processing Workflow of Formalin-Fixed Paraffin-Embedded (FFPE) Tissue Yielding up to 10,000 Proteins per Sample
Presented by Moe Haines,
Broad Institute of MIT and Harvard, Cambridge, MA 

• WP 765
• Wednesday, June 5, 10:30-11:30, 12:30-2:30

High-Throughput and Automated Cellular and Tissue Lysis using COVARIS Acoustic Technology for Proteomics
Presented by Josselin Ortiz,
Cedars-Sinai Medical Center, Beverly Hills, CA 

• WP 503
• Wednesday, June 5, 10:30-11:30, 12:30-2:30

Leveraging the Covaris AFA-Sonication to Develop a High-Throughput Assay for Deep Proteome Coverage of Fresh Frozen Tissue Sections​​​​​​​
Presented by Saeed Seyedmohammad,
Cedars-Sinai Medical Institute, Los Angeles, CA   

• WP 774
• Wednesday, June 5, 10:30-11:30, 12:30-2:30