Proteomic Workflows with High-Throughput Protein Digestion

Proteomics is an essential field for understanding biological processes and systems, with protein digestion forming a critical step in its workflows. Traditionally, protein digestion is carried out in solution using proteolytic enzymes like trypsin, which specifically hydrolyzes peptide bonds at lysine and arginine residues. This process generates peptides well-suited for ionization and fragmentation in mass spectrometry (MS) applications. However, conventional digestion methods require 12–24 hours to achieve completion, creating significant bottlenecks in proteomic workflows.
Ultrasonication offers a powerful alternative, dramatically shortening digestion times from hours to just a few minutes. When combined with advanced multi-sample sonicators such as the Hielscher CupHorn, VialTweeter, and the 96-well plate sonicator UIP400MTP, ultrasonication enables accelerated, high-throughput proteomics. These technologies streamline workflows, reducing sample preparation time and increasing efficiency without compromising reproducibility or data quality.

The Role of Ultrasonic Energy in Protein Digestion

Ultrasonication employs focused ultrasound waves to create cavitation—localized microbubbles that collapse to generate intense shear forces. This phenomenon enhances mass transfer, promotes the mixing of enzymes with substrates, and unfolds protein structures, exposing cleavage sites to proteolytic enzymes like trypsin.
The result? A significant reduction in digestion time without compromising efficiency or reproducibility.

Ultrasonic-Enhanced Proteolytic Digestion: Methodology and Results

Accelerated Digestion Protocol

Ultrasonic-assisted digestion combines proteolytic enzymes and ultrasonic energy to expedite workflows. For example, using the Hielscher UP200St-CupHorn (200 W, 26 kHz), the digestion workflow proceeds as follows:

1. Reduction: Protein samples (0.5 mg/mL, 20 µL) are treated with DTT (2 µL, 110 mM) in ammonium bicarbonate buffer (12.5 mM). Sonication is applied at 50% amplitude for 5 minutes.
2. Alkylation: After adding IAA (2 µL, 400 mM), sonication is repeated under the same conditions.
3. Digestion: Samples are diluted and incubated with immobilized trypsin nanoparticles. A final round of sonication (5 minutes) completes the digestion. Peptides are separated, dried, and stored for MS analysis.

This ultrasonic method reduces total preparation time from 12 hours to under 30 minutes. Despite the accelerated process, peptide yield and quality remain consistent with traditional overnight methods.

Efficiency of Ultrasonic Protein Digestion

In comparative studies using E. coli proteomes:

• Protein Identification: Ultrasonically digested samples identified 777 proteins in 5 minutes, compared to 817 in 12 hours. Shared protein identifications exceeded 70%.
• Reproducibility: Replicate analyses showed correlation values above 98% within each method, demonstrating reliability.
• Selectivity: Certain proteins were preferentially digested by each method, with ultrasonic digestion favoring 65 proteins and overnight digestion 54 proteins. Such nuanced differences underline the unique potential of ultrasonication for specific applications.

Label-free protein quantification results of seven spiked proteins into an E. coli
sample. The following proteins were added at two different levels as noted in the column
named as “Theo Ratio”. Theo Ratio is the theoretical ratio between the two levels used in this
experiment. Bovine serum albumin (ALBU), β-lactoglobulin (LACB), α-S1 casein (CASA1),
α-S2 casein (CASA2), cytochrome c (CYC), ovalbumin (OVAL) and carbonic anhydrase 2
(CAH2). Ratios were calculated using LFQ protein intensities obtained from MaxQuant
analysis. The student’s t test was applied to compare the values obtained with each method (p>0.01, n=3, t-theoretical=4.6).
Study and graphic: © Martins et al., 2019)

Nanoparticle-Immobilized Trypsin

The integration of immobilized trypsin nanoparticles (e.g., T-FMNPs) with ultrasonication further enhances proteomics workflows. These nanoparticles provide a high surface area for enzyme-substrate interactions, increasing efficiency. When applied to complex proteomes, such as E. coli, the combined method achieves:

• Speed: Complete digestion within 5 minutes.
• Accuracy: Comparable protein quantification to traditional methods (p > 0.01, n=3).
• Scalability: Adaptation to multi-well platforms like the UIP400MTP enables high-throughput processing.

Click here for the detailed protocol with step-by-step instructions!
(cf. Martins et al., 2019)

Proteolytic digestion at high-speed and high-throughput with Hielscher sonicators

The Best Sonicator Models for Proteomics

Hielscher Ultrasonics offers various sonicator models for simultaneous multi-sample preparation facilitating high-throughput workflows. Whether you work with vials, test tubes, multi-well plates (e.g. 6-, 24-, 96-well plates) or Petri dishes – we offer you the ideal sonicators for your experiments.

UIP400MTP Multi-Well Plate Sonicator

For ultimate throughput, the UIP400MTP provides the capability to process 96-well plates ultrasonically. Compatible with any standard microplate, the UIP400MTP does not require expensive proprietary disposables and gives you the freedom to choose the best multi-well plate for your research. By delivering uniform energy across the plate, it enables rapid reduction, alkylation, and digestion of up to 200 complex proteomes in just 1 hour. This level of automation and efficiency is crucial for high-throughput proteomics and clinical applications. Learn more about the multi-well plate sonicator!

VialTweeter

The VialTweeter is tailored for laboratories requiring the simultaneous sonication of up to 10 vials or test tubes. Its non-invasive approach eliminates cross-contamination risks while ensuring reproducible protein digestion. This device is ideal for researchers working with limited sample volumes or diverse sample types.
Learn more about the VialTweeter multi-tube sonicator!

Hielscher UP200St-CupHorn

The CupHorn sonicator is a powerful device designed for simultaneous processing of multiple samples in sealed containers. It ensures uniform ultrasonic energy distribution and precise temperature control. The ability to process up to five samples simultaneously, coupled with its compatibility with reduced, alkylated, and digested workflows, makes the CupHorn a reliable tool for MS-based proteomics.
Learn more about the CupHorn SonoReactor!

Step-by-Step Protocol for Ultrasonication-Assisted Proteolytic Digestion Using Nanoparticle-Immobilized Trypsin

This protocol by Martins et al. (2019) is optimized for rapid protein digestion using ultrasonic energy and nanoparticle-immobilized trypsin (T-FMNPs). The outlined steps ensure efficient reduction, alkylation, and proteolysis suitable for mass spectrometry (MS) applications.
Protocol Steps

1. Reduction of Disulfide Bonds
   • Add 2 µL of DTT solution (110 mM) to the 20 µL protein sample (0.5 mg/mL) in AmBic buffer.
   • Place the sample tube in the sonoreactor UP200St-CupHorn.
   • Sonicate the sample for 2.5 minutes at 50% amplitude (200 W, 26 kHz).
   • Pause for a brief interval to allow cooling, then sonicate for another 2.5 minutes under the same conditions.
2. Alkylation of Reduced Cysteine Residues
   • Add 2 µL of IAA solution (400 mM) to the reduced protein sample.
   • Sonicate for 2.5 minutes at 50% amplitude to facilitate alkylation.
   • Pause for cooling, then sonicate for an additional 2.5 minutes.

   Note: Minimize exposure of the alkylated sample to light to prevent IAA degradation.

3. Sample Dilution
   • Dilute the alkylated protein sample to a final volume of 100 µL using 25 mM AmBic buffer containing 4% acetonitrile (v/v).
   • Mix thoroughly by gentle pipetting.
4. Proteolytic Digestion with Nanoparticle-Immobilized Trypsin
   • Add 20 µL of T-FMNP solution (3 mg/mL) to the diluted protein sample.
   • Sonicate the mixture in the sonoreactor for 2.5 minutes at 50% amplitude.
   • Pause for cooling, then sonicate for another 2.5 minutes under the same conditions.
5. Separation of Trypsin Nanoparticles
   • Use a magnet to separate the T-FMNPs from the supernatant containing digested peptides.
   • Transfer the supernatant to a new microcentrifuge tube.
6. Peptide Preparation for MS Analysis
   • Dry the supernatant containing peptides in a vacuum centrifuge.
   • Store the dried peptides at -20°C until further analysis by mass spectrometry.