Large Scale EasyPep MS Sample Preparation for Phosphopeptide Enrichment Workflows

Importance of the topic

Phosphorylation is a key post-translational modification that regulates protein function in signaling networks. Mass spectrometry-based phosphoproteomics demands enrichment of low-abundance phosphopeptides from complex digests. A rapid, scalable, and reproducible sample preparation workflow is essential to support high-throughput studies in basic research and clinical applications.

Objectives and study overview

This work evaluates the scalability and performance of the EasyPep™ sample preparation chemistry for phosphopeptide enrichment from large-scale protein digests (>1 mg). The study benchmarks small-scale spin columns against a newly developed large-scale column format and compares the workflow to conventional protocols. Compatibility with different cell lines, human plasma, and isobaric labeling reagents (TMT 11-plex and TMTpro 16-plex) is assessed.

Methodology

• Cell lysis in denaturing buffer with phosphatase inhibitors and nuclease treatment to reduce viscosity
• Combined reduction/alkylation at 95 °C followed by digestion with trypsin/Lys-C protease mix
• Peptide clean-up using mixed-mode spin columns (10–100 μg) or large-scale vacuum columns (>1 mg resin)
• Fe-NTA IMAC enrichment of phosphopeptides
• High-pH reversed-phase fractionation prior to LC-MS
• Isobaric labeling with TMT 11-plex or TMTpro 16-plex reagents

Used instrumentation

• Thermo Dionex Ultimate 3000 Nano LC system with 50 cm EASY-Spray™ C18 column
• Thermo Q Exactive™ Plus Hybrid Quadrupole-Orbitrap mass spectrometer
• Thermo Orbitrap Fusion™ mass spectrometer for TMT-labeled samples
• Pierce™ Rapid Gold BCA and Quantitative Colorimetric Peptide Assay kits
• Pierce™ Hi-Select™ Fe-NTA phosphopeptide enrichment kit

Main results and discussion

• Large-scale column format produced ~4,800 protein IDs and ~28,800 peptide IDs, matching small-scale spin performance.
• Phosphopeptide specificity ≥95%, cysteine alkylation efficiency ~99%, zero missed cleavages ~84–87%.
• Sample prep time of 2 hours for 10–100 μg and 3–5 hours for >1 mg, significantly shorter than conventional workflows.
• TMT 11-plex and TMTpro 16-plex yielded similar total protein and peptide identifications, with slightly fewer quantified phosphopeptides for TMTpro.
• Consistent results observed in HeLa S3, A549 cell lines and human plasma digests.

Benefits and practical applications

• Streamlined, hands-on workflow with minimal variability between labs
• Scalable from microgram to milligram input amounts
• Compatibility with multiplexed quantitative proteomics
• Applicable to diverse biological samples including cells, tissues, serum and plasma

Future trends and possible applications

• Further optimization of TMTpro-based phosphoproteome analyses to enhance coverage
• Automation of the EasyPep workflow for high-throughput clinical studies
• Extension to other post-translational modifications using tailored enrichment chemistries
• Integration with multi-omics platforms for systems biology investigations

Conclusion

The EasyPep sample preparation kit delivers a rapid, robust, and scalable workflow for phosphopeptide enrichment from large protein digests. It matches or exceeds conventional methods in yield, specificity and reproducibility while drastically reducing processing time and hands-on effort. Compatibility with isobaric labeling and multiple sample types makes it a versatile tool for quantitative phosphoproteomics.

Reference

• Bomgarden R.D., Flora A., Snovida S., Jensen P., Rogers J.C. Large Scale EasyPep MS Sample Preparation for Phosphopeptide Enrichment Workflows. Thermo Fisher Scientific, 2019.