EasyPep Sample Preparation Technology for Rapid and Efficient Mass Spectrometry-based Proteomics

Significance of the Topic

Advances in mass spectrometry instrumentation have unlocked high-resolution analysis of complex proteomes, yet upstream protein sample preparation remains a bottleneck due to lengthy protocols, variable yields, and low reproducibility. Standardizing and accelerating sample preparation workflows is critical for applications ranging from biomarker discovery to clinical QA/QC and large-scale multi-omics studies.

Objectives and Study Overview

This study introduces two new formats within the Thermo Scientific™ EasyPep™ sample preparation technology:

• A Maxi kit for large-scale processing (0.5–2 mg protein) tailored to post-translational modification analysis.
• A 96-well plate format for high-throughput, automated workflows on 10–100 µg protein samples.

The work compares all three EasyPep formats (Mini, Maxi, 96-well) across diverse sample types—including human cell lines, plasma/serum, yeast, E. coli, tissues, and FFPE sections—to assess performance, reproducibility, and compatibility with TMT labeling and phosphopeptide enrichment.

Methodology and Instrumentation Used

Sample Preparation:

• Cell and tissue lysis in buffer with reduction, alkylation, and Trypsin/Lys-C digestion (under 3–4 hr).
• Peptide cleanup via Mini/Maxi columns or 96-well filter plates under centrifugation, vacuum, or positive-pressure modes.
• Optional plasma depletion with Top14 spin columns.
• Phosphopeptide enrichment using Fe-NTA resins and TMT6plex labeling before or after digestion.

Instrumentation:

• Thermo Scientific™ Dionex™ UltiMate™ 3000 Nano LC with 50 cm EASY-Spray C18 column.
• Thermo Scientific™ Q Exactive™ Plus Hybrid Quadrupole-Orbitrap mass spectrometer.
• Automated handling on Hamilton™ STARlet™ liquid handler for the 96-well format.

Main Results and Discussion

Peptide and Protein Identifications:

• All formats yielded comparable identifications from HeLa and A549 cells (≈5 200 proteins, >30 000 peptides) within a <4-hr workflow.
• EasyPep outperformed conventional protocols by 10–20 % more IDs and reduced missed cleavages; TMT labeling efficiency exceeded 99 % with near–zero missed modifications.

Sample-Type Compatibility:

• High reproducibility (CVs <1 %) across cell lines, plasma/serum (depleted and non-depleted), yeast, bacteria, and fresh/fixed tissues (CVs <5 %).
• FFPE and fresh-frozen tissues yielded similar identifications (~29 000–31 000 peptides) after xylene/ethanol deparaffinization.

Phosphoproteomics and PTM Analysis:

• Fe-NTA enrichment post-EasyPep gave >95 % phosphopeptide specificity within 3–5 hr, matching conventional workflows.
• Maxi format optimized for PTM workflows on milligram-scale inputs.

Benefits and Practical Applications

The EasyPep technology delivers:

• Rapid, streamlined sample preparation (<4 hr) with standardized protocols.
• Scalable formats accommodating both low-microgram and milligram inputs.
• Seamless integration with isobaric labeling and phosphopeptide enrichment for quantitative and PTM studies.
• Compatibility with automation platforms for high-throughput screening and clinical testing.
• Consistent high yields and identification rates conducive to biomarker discovery, QA/QC, and drug development pipelines.

Future Trends and Possibilities

As proteomics moves toward larger cohort studies and multi-omics integration, automated and miniaturized workflows will be essential. Potential developments include:

• Microfluidic implementations to further reduce input and reagent consumption.
• Enhanced multiplexing strategies using novel isobaric tags beyond TMT6plex.
• AI-driven protocol optimization to tailor sample prep to unique matrix challenges.
• Cloud-based data processing integration to link upstream prep metadata with downstream analytics.

Conclusion

Expanding the EasyPep sample preparation portfolio with Maxi and 96-well formats addresses the dual needs of high throughput and large-scale PTM workflows. The standardized chemistry and automation compatibility yield rapid, reproducible, and high-quality proteomic datasets across diverse sample types, positioning EasyPep as a versatile solution for research and clinical proteomics.