Automation for LC/MS Sample Preparation: High Throughput In-Solution Digestion and Peptide Cleanup Enabled by the Agilent AssayMAP Bravo Platform

Significance of the Topic

The efficiency and reproducibility of protein and peptide sample preparation are critical for quantitative LC/MS proteomics. Traditional manual workflows are labor-intensive, poorly scalable, and prone to variability, limiting throughput and method transfer between laboratories. Automated platforms designed specifically for in-solution digestion and peptide cleanup can address these challenges, enabling high-throughput biomarker discovery and routine QC/QA applications in clinical and industrial research.

Objectives and Study Overview

This application note evaluates the Agilent AssayMAP Bravo platform for automating in-solution digestion and reversed-phase peptide cleanup. The goals were to demonstrate:

• Day-to-day and plate-to-plate reproducibility of multiplexed digestion of bovine serum albumin (BSA).
• Peptide recovery and cleanup performance using C18 and RP-S AssayMAP cartridges.
• Load capacity and analyte bias across a range of peptide hydrophobicities and digest load masses.

Methodology and Instrumentation

The automated workflow consisted of two user-customizable protocols on the AssayMAP Bravo:

1. In-Solution Digestion: Parallel processing of up to four 96-well plates, with denaturation (urea or guanidine), reduction, alkylation, and tryptic digestion at controlled temperatures.
2. Peptide Cleanup: High-throughput reversed-phase cleanup using disposable C18 or RP-S microchromatography cartridges for desalting and concentration.

Key instrumentation and consumables included:

• Agilent AssayMAP Bravo liquid handler with Peltier thermal station and disposable 96-channel cartridges.
• Agilent PlateLoc thermal sealer for incubation steps.
• Agilent 6550 iFunnel QTOF LC/MS coupled to a 1290 Infinity UHPLC with AdvanceBio PepMap columns.

Main Results and Discussion

Reproducibility of digestion and cleanup was assessed by monitoring 25 BSA peptides across 128 samples over two days. Using guanidine-based denaturation, average peptide peak area coefficients of variation (%CV) were below 2.6 % for both days. Urea-based denaturation yielded slightly higher %CVs (<3.7 %).
Peptide recovery versus online LC cleanup demonstrated>90 % recovery and <2.5 % CV for peptides of low-to-moderate hydrophobicity at loads from 3 to 75 µg. Highly hydrophobic peptides showed reduced recovery at low load masses but remained reproducible. Load capacity studies indicated stable performance for up to 100 µg BSA digest on both cartridge types, with hydrophilic peptides becoming displaced beyond that point.

Benefits and Practical Applications of the Method

The AssayMAP Bravo platform delivers:

• High throughput: Simultaneous digestion of up to 384 samples and cleanup of 96 samples.
• Reproducible results: Day-to-day %CVs consistently below 5 % for 25 monitored peptides.
• Scalability and portability: Simple protocol customization and minimal hands-on time facilitate method transfer and scale-up.

These attributes support large-scale biomarker studies, routine proteomic QC/QA, and clinical research requiring robust sample preparation.

Future Trends and Opportunities

Further development may include integration of stable isotope labeling, on-deck enrichment workflows, and advanced multiplexing with isobaric tags. Expansion of cartridge chemistries could enable targeted enrichment of post-translational modifications. Coupling with machine-learning-driven protocol optimization will accelerate method development and ensure consistent performance across diverse sample types.

Conclusion

The Agilent AssayMAP Bravo platform streamlines LC/MS sample preparation by automating in-solution digestion and peptide cleanup, achieving high reproducibility, broad dynamic range, and scalable throughput. This technology addresses key bottlenecks in quantitative proteomics and supports large-scale biomarker discovery and routine analytical applications.

References

1. Spicer V, et al. Sequence-specific retention calculator. A family of peptide retention time prediction algorithms in reversed-phase HPLC: applicability to various chromatographic conditions and columns. Anal. Chem. 2007, 79(22):8762–8768.