Leveraging AFA Ultrasonication as a Versatile Sample Preparation Tool for Proteomics Core Facilities

During ASMS 2023, Jennifer Roof, a Research Technician with the CHOP-Penn Proteomics Core Facility at the Children’s Hospital of Philadelphia, University of Pennsylvania, shared how Covaris’ R230 Focused-ultrasonicator, powered by Adaptive Focused Acoustics® (AFA®) technology, has been a game-changer for their sample preparation workflow as a core facility by providing a flexible, efficient and reproducible method.

Introduction

Proteomics is a fascinating yet complex field, with numerous techniques used to understand the biological functions of proteins. One standout technique that has gained attention is bottom-up proteomics, a process in which proteins are extracted from a range of sources, digested into peptides, and then analyzed using LC-MS systems. The CHOP-Penn team has mastered this process, with AFA technology playing an essential role in their workflow for protein disruption, lysis, extraction, purification, & digestion at their core facility by providing a method that is flexible, efficient and reproducible.

The CHOP-Penn facility handles an extensive range of samples – from formalin-fixed paraffin-embedded (FFPE) to extracellular matrix, cells, tissues, and even species like mice, monkeys, and parasites. Given such diversity, a robust method for protein extraction is needed. Yet, finding a robust method that is able to reproducibly extract proteins from every sample type can be challenging, with many variables to consider. This leads to several questions: Which buffer is best? How many samples need to be processed? What is the volume of each sample?

Three Pillars to Protein Sample Prep Success: Flexibility, Efficiency and Reproducibility

In this process, three factors are key: flexibility, efficiency, and reproducibility. So, how does Covaris’ R230 Focused-ultrasonicator contribute to achieving these principles? Let’s take a look:

Efficiency:

The R230 Focused-ultrasonicator provides:

  • Complete protein extraction, particularly from challenging samples
  • Improved homogenization of samples
  • DNA shearing, which negates the need for Benzonase
  • Fast results, processing an entire 96-well plate in just 10 minutes per column

Flexibility:

The R230 Focused-ultrasonicator provides:

  • Functionality at both low and high temperatures
  • Optimizable settings for various sample types and buffers
  • Compatibility with many downstream workflows, because you can do it in a 96-well plate

Reproducibility:

The R230 Focused-ultrasonicator provides:

  • High throughput 96-well plate format
  • Less hands-on time
  • Compatibility with various automation platforms

Case Study: Examining the Efficiency of Protein Extraction Using the R230 Focused-ultrasonicator

The team conducted an experiment using a standard protein quantification method (Coomassie Stain: in-gel Bradford) on Cryptosporidium parvum oocysts and standard splenocytes (Figure 1). Data revealed protein extraction efficiency before and after R230 processing. The results clearly showed that the use of Covaris’ R230 Focused-ultrasonicator led to improved protein extraction.

 

Figure 1. Protein extraction efficiency in oocysts and splenocytes: before and after R230 processing.

They then repeated the experiment with oocysts, using replicates of 5 million and 1 million. Again, it was observed that protein extraction after processing in the R230 Focused-ultrasonicator significantly outperformed the extraction before processing (Figure 2). Across all samples, the team had similar intensities and significant protein ID overlap. A total of 1,929 proteins were identified across all 15 samples, out of 2,000 proteins identified total, demonstrating excellent reproducibility (Figure 3).

Figure 2. Protein extraction efficiency: C. parvum Oocysts using replicates of 5 million and 1 million before and after R230 processing

Figure 3. Reproducibility: C. parvum extraction

The R230 Focused-ultrasonicator bead homogenization capability is a key feature that is particularly useful in SP3 workflows with magnetic beads. Often, these beads can adhere to well bottoms or sides, making homogenization challenging. However, R230 sonication significantly improved this process.

The team conducted an experiment in which they homogenized the beads in half of the wells, while leaving the others untouched. Data showed an increase in precursor IDs and protein IDs when using R230 processing compared to gray water bath sonication (Figure 4). Data also indicated that the coefficient of variations was reduced with R230 ultrasonication, even at lower cell inputs (Figure 5).

Figure 4. Precursor and protein IDs

Figure 5. Reproducibility: Coefficient of variation.

Moreover, the team explored the FFPE workflow and discovered that the R230 Focused-ultrasonicator excels at homogenizing for FFPE, and the overall processing time is fast, as depicted in Figure 6.

Figure 6. AFA settings

Showcasing the Flexibility of Protein Extraction Using the R230 Focused-ultrasonicator

The R230 Focused-ultrasonicator provides flexible protein extraction due to its adjustable temperature settings, making it compatible with various buffers and sample types. The water bath’s temperature can be set anywhere between 5 to 40 degrees Celsius, allowing for methods at 10 degrees with urea or at room temperature. However, sonicating SDS at low temperatures led to unexpected polymerization on tube walls, making it hard to remove. Therefore, it’s recommended to avoid sonicating SDS at 10 degrees.

Additionally, the R230 Focused-ultrasonicator features fully customizable settings to optimize for different samples and buffers. Parameters such as duty factor and cycles per burst can be adjusted based on user needs, which is especially useful for core facilities managing projects from start to finish.

Highlighting Protein Extraction Reproducibility Using the R230 Focused-ultrasonicator

The R230 Focused-ultrasonicator also streamlines workflows by reducing sample transfers, thus minimizing loss and pipetting errors. It enables workflows that require fewer tools, leading to less batch-to-batch variability. Moreover, it’s compatible with both automated and semi-automated processes.

In addition, the R230 Focused-ultrasonicator is compatible with various digestion and cleanup methods such as iST, S-Trap, SP3, and EvoTips. It works well with different temperature control solutions, homogenization tools, and is directly plate-compatible for in-solution digests. While it doesn’t entirely replace other tools, the R230 offers temperature control, lessening the need for a thermomixer unless higher temperatures are required. It’s also compatible with the PreOmics Beatbox for sample homogenization and can handle tissue directly.

Its 96-well plate design facilitates easy transfer to an iST setup, S-Trap, or EvoTips, minimizing the number of tools used and sample transfers, aiding reproducibility and reducing batch-to-batch variability.

The R230 Focused-ultrasonicator also offers optimization for challenging samples. It has been successfully used on a range of sample types such as T cells, ECM, neurons, and different tissues, underlining its reproducibility.

Other features include DNA shearing, which reduces sample viscosity and prevents contamination, eliminating the need for Benzonase.

Conclusion

For facilities that need to handle a wide range of sample types, identifying a method that extracts proteins from every sample completely and reliably is challenging. This technique must be flexible, efficient, and reproducible. Covaris’ R230 Focused-ultrasonicator, powered by AFA technology, has stepped up to the task, proving to be a transformative tool for core facilities and their sample preparation.

Interested in how the R230 Focused-ultrasonicator could transform your operations with its flexibility, efficiency, and reproducibility? Find out more on the R230 product page.