Best Practices for Protein Analysis with the Agilent ProteoAnalyzer System

Importance of Topic

Reliable and reproducible protein quality control underpins workflows in biopharmaceutical development, academic research, and industrial analytics. Conventional SDS-PAGE methods consume hours of manual work and large sample volumes, limiting throughput and consistency. Automated capillary electrophoresis with SDS (CE-SDS) on platforms such as the Agilent ProteoAnalyzer system addresses these limitations by enabling parallel analysis of multiple samples with high resolution, sensitivity, and minimal hands-on time.

Objectives and Study Overview

This technical overview defines best practices for protein sizing, purity assessment, and quantification using the Agilent ProteoAnalyzer with the Protein Broad Range P240 kit. It examines critical parameters including sample buffer compatibility, injection conditions, labeling temperature, plate handling, and data processing, and compares results to established reference materials to ensure accuracy and precision across a broad range of protein types and concentrations.

Used Instrumentation

• Agilent ProteoAnalyzer system with 12-capillary array
• Protein Broad Range P240 kit, low and optional upper marker workflow
• Agilent ProSize data analysis software for electropherogram and digital gel visualization

Methods and Instrumentation

Protein samples including BSA and NISTmAb were prepared under reduced and non-reduced conditions using the kit’s rapid fluorescent labeling protocol. Samples and ladders were analyzed via two methods: lower marker only (LM-only) and combined lower and upper marker (LM+UM). Buffer compatibility was assessed by diluting samples into PBS, high-salt solutions, and detergent buffers and comparing migration profiles. Injection voltage/time, incubation temperature (70 °C vs 85 °C), and plate storage (room temperature vs 4 °C) were systematically varied. ProSize software peak detection thresholds and integration parameters were adjusted to optimize quantification.

Main Results and Discussion

• Buffer effects: Samples and ladders must be prepared in identical buffers to avoid sizing errors. High salt and detergents shift migration times and apparent molecular weights by up to 20% when mixed mismatched. LM+UM alignment reduced sizing error to below 10% across buffers.
• Injection optimization: A 10-second injection generated peak saturation (>80 000 RFU) and triggered software warnings. Reducing injection to 6 seconds maintained peak heights near 50 000 RFU for optimal linearity.
• Labeling temperature: Incubation at 70 °C improved resolution between heavy and nonglycosylated chains, yielded monomeric purity and glycan occupancy values matching NIST Reference Material, and lowered variability compared to 85 °C.
• Plate capacity and stability: Up to three 96-well plates (288 samples) can be loaded consecutively without user intervention. Size, concentration, and peak height of ladder fragments remained stable over 24 sequential runs (CV <3%).
• Sample storage: Labeled samples showed significant peak broadening and intensity loss after 3 days at room temperature or 4 °C. For accurate results, run samples within 24 hours of labeling.
• Data analysis: ProSize software provides electropherogram overlays, digital gel images, and quantitative peak tables. Customizable peak width and height thresholds improve integration of low-abundance impurities and smears.

Benefits and Practical Use

• High throughput: Parallel analysis of 12 samples per run reduces overall assay time to under 1 hour.
• Automation and consistency: Automated capillary cleaning and gel replenishment deliver reproducible separations across diverse protein samples.
• Wide dynamic range: Sensitive CCD detection offers over three orders of magnitude in linear response for trace impurities.
• Scalable workflow: Compatible with purified antibodies, membrane proteins, fermentation broths, and crude lysates, supporting R&D and QC laboratories.
• Simplified data management: Integrated software enables rapid decision making and easy archiving of results.

Future Trends and Potential Uses

• Integration with laboratory information management systems (LIMS) for real-time QC dashboards.
• Advanced multiplexed assays combining CE-SDS with on-line mass spectrometry or fluorescence lifetime measurements.
• Machine learning algorithms for automated peak annotation and impurity classification.
• Expansion of marker kits to cover broader mass ranges and post-translational modification analyses.
• Miniaturized disposable capillary cartridges to reduce maintenance and cross-contamination risks.

Conclusion

The Agilent ProteoAnalyzer system with the Protein Broad Range P240 kit provides a robust, automated platform for fast and precise protein sizing, purity assessment, and quantification. By following best practices in buffer matching, injection settings, labeling temperature, and data analysis, laboratories can achieve reproducible results that align with NIST standards and meet the demands of high-throughput biotherapeutic development and industrial QC.

Reference

1. Agilent Technologies. Protein Sizing and Quantification with the Agilent ProteoAnalyzer System; Technical Overview, 2023; Publication 5994-6718EN.
2. Agilent Technologies. Quality Analysis Using the Agilent ProteoAnalyzer System and SDS-PAGE: A comparison of sizing and quantification performance; Technical Overview, 2023; Publication 5994-6934EN.
3. Agilent Technologies. Agilent Protein Broad Range P240 Kit Quick Guide for the ProteoAnalyzer System; Kit Quick Guide, 2024; Publication D0031125.
4. National Institute of Standards and Technology; U.S. Department of Commerce. Reference Material 8671: NISTmAb, Humanized IgG1κ Monoclonal Antibody, Lot 14HB-D-002; Reference Material Information Sheet, 2023.
5. Agilent Technologies. Analysis of NIST Antibody on the Agilent ProteoAnalyzer System; Technical Overview, 2023; Publication 5994-6960EN.
6. Turner A.; Yandrofski K.; Telikepalli S.; King J.; Heckert A.; Filliben J.; Ripple D.; Schiel J.E. Development of Orthogonal NISTmAb Size Heterogeneity Control Methods. Anal. Bioanal. Chem. 2018, 410(8), 2095–2110.
7. Agilent Technologies. ProSize Data Analysis Software for Protein; User Manual, 2023; Publication D0033432.