Efficient proteomics with an automated sample preparation strategy leveraged by Evotip Pure and the AssayMAP Bravo

Importance of the topic

Proteomics has rapidly evolved into a high-throughput discipline, driven by advances in high-end mass spectrometry and automation. Sample preparation has become the main bottleneck in modern proteomic workflows, with demand for robust, reproducible, and scalable pipelines.

Objectives and overview of the study

This application note describes a fully automated proteomic sample preparation strategy combining Protein Aggregation Capture (PAC) digestion with in-line Evotip Pure loading on an Agilent AssayMAP Bravo. The workflow is designed for up to 96 samples per run and scales to 480 samples per day, offering flexibility in digestion time (30 min to overnight) for throughput versus depth trade-off.

Methods and instrumentation

HeLa cell lysates were processed in boiling 5% SDS and subjected to PAC on magnetic hydroxyl microparticles. A single isopropanol wash removed contaminants, followed by combined Lys-C and trypsin digestion (1:100 and 1:25 enzyme-to-protein ratios) at 37 °C with agitation under a sealed lid. Digestion times ranged from 30 min to 16 h. Peptides were transferred directly onto Evotip Pure tips using a layered “sandwich” loading on the AssayMAP Bravo 96-head and then analyzed on Evosep One coupled to timsTOF HT (DIA-PASEF) or a QQQ 6495C (dynamic MRM). Data were processed with DIA-NN in library-free mode and Skyline for targeted assays.

Instrumentation

• Evotip Pure (Evosep)
• Agilent AssayMAP Bravo liquid handling robot
• Evosep One LC system
• timsTOF HT mass spectrometer (Bruker)
• Aurora Elite and Rapid75 columns (IonOpticks)
• QQQ 6495C triple quadrupole (Agilent)

Main results and discussion

Digestion efficiency increased with time, yielding 89% fully cleaved peptides after 16 h (CV ~5%). Short digestions of 2 h and 30 min achieved 73% and 65% efficiency with CVs <10% and ~7%, respectively, demonstrating high reproducibility. Peptide-specific kinetics highlighted the need to tailor digestion times for certain targets.

Robustness was validated by preparing 480 samples in one day (30 min digestion), with mean efficiency of 67% across five plates (CV <7%) and low intra- and inter-plate variability (PCA analysis).

Sensitivity assessment using 5–1000 ng HeLa digest showed identification of ~8 000 precursors and 2 000 proteins from 5 ng input and >90 000 precursors and 8 000 proteins from 1000 ng (Whisper Zoom 40 SPD). Quantitative precision was excellent, with protein-level CVs <20% at 10 ng and ~6-7% at 1000 ng.

Benefits and practical applications

• High throughput: up to 500 samples per day depending on digestion time
• Flexible depth: adjustable digestion time balances speed and completeness
• Reproducibility: low CVs across hundreds of samples and plates
• Sensitivity: deep proteome coverage from low nanogram inputs
• Seamless integration: direct peptide loading onto Evotip for LC-MS

Future trends and potential applications

Integration with additional sample preparation modules such as post-digestion enrichment, isobaric labeling, or phosphopeptide capture could extend the platform’s versatility. Clinical and industrial QC proteomics stand to benefit from the robustness and throughput. Further miniaturization and adaptation to other robotic platforms will expand accessibility.

Conclusion

An end-to-end automated PAC digestion and Evotip loading workflow on the AssayMAP Bravo offers a rapid, scalable, and sensitive solution for high-throughput proteomics. It delivers reproducible digestion, deep proteome coverage from low inputs, and seamless coupling to LC-MS analysis.

Reference

1. Batth TS, Tollenaere M, Rüther P, Gonzalez-Franquesa A, Prabhakar BS, Bekker-Jensen S, Deshmukh AS, Olsen JV (2019) Protein Aggregation Capture on Microparticles Enables Multipurpose Proteomics Sample Preparation. Mol Cell Proteomics. mcp.TIR118.001270