Proteomics workflow solutions

Significance of the Topic

Proteomics is central to understanding protein composition, structure, and function in biological systems. Robust, reproducible, and high-resolution separations underpin reliable identification and quantitation of peptides and intact proteins across diverse applications from fundamental research to biopharmaceutical development.

Study Objectives and Overview

This work presents integrated workflows for bottom-up, middle-down, and top-down proteomics, combining advanced chromatographic columns, consumables, and mass spectrometry platforms. The goal is to demonstrate enhanced performance metrics such as reproducibility, throughput, and sensitivity through tailored solutions spanning sample preparation to data acquisition.

Applied Instrumentation

• Vanquish Neo UHPLC system for high-pressure, nano and capillary flow separations
• Orbitrap Exploris 480 mass spectrometer with EASY-Spray ion source
• Chromatography columns: EASY-Spray PepMap Neo, µPAC HPLC, MAbPac RP capillary
• Trap columns: Acclaim PepMap 100 C18 and µPAC trapping columns
• Emitters: EASY-Spray nano and capillary configurations, µPAC compatible emitters
• Protein digestion: SMART Digest Proteinase K kits, automated AccelerOme platform
• Sample vessels and sealing: SureSTART vials and caps, WebSeal Plate+ well plates and tapes

Main Results and Discussion

Bottom-up peptide mapping using EASY-Spray PepMap Neo columns and Vanquish Neo UHPLC achieved outstanding retention time stability over 1,600 injections spanning 176 days, demonstrating exceptional robustness for long-term studies. µPAC HPLC columns enabled high-throughput analyses with 20–90 minute gradients at 300–1000 nL/min, maintaining peak capacities and deep proteome coverage suitable for routine and complex sample profiling. Middle-down experiments with MAbPac RP capillary columns delivered sensitive monitoring of glycosylation site occupancy and deamidation, leveraging capillary flow for enhanced signal and fast separations. Top-down intact mass measurements on polymer-based MAbPac RP columns reduced carry-over below 1 % while preserving sharp peak shapes and column longevity under harsh wash conditions.

Benefits and Practical Applications

These cohesive workflows provide:

• High reproducibility and minimal column-to-column variability
• Extended lifetimes and low carry-over for large biomolecules
• Scalable throughput from discovery proteomics to biopharma QA/QC
• Simplified connectivity via click-and-spray emitters and integrated consumables

Applications range from deep proteome profiling and post-translational modification analysis to intact therapeutic protein characterization and automated sample preparation in regulated environments.

Future Trends and Opportunities

Advancements may include:

• Integration of real-time data analytics and AI-driven method optimization
• Automated end-to-end sample handling with robotic platforms
• High-throughput single-cell and spatial proteomics workflows
• Miniaturized microfluidic systems for rapid diagnostic assays

These developments promise faster, deeper, and more accessible proteomic insights across research and clinical settings.

Conclusion

The described workflow solutions combine state-of-the-art chromatography, consumables, and mass spectrometry to deliver reliable, high-performance proteomics across bottom-up, middle-down, and top-down applications, empowering researchers to achieve consistent, high-quality results.