Automated, High Precision Tryptic Digestion and SISCAPA-MS Quantification of Human Plasma Proteins Using the Agilent Bravo Automated Liquid Handling Platform

Significance of the Topic

Automated, high-precision tryptic digestion coupled with SISCAPA-MS quantification addresses the need for reliable and scalable measurement of low-abundance plasma proteins. Such workflows support clinical biomarker verification, large-scale proteomic studies, and quality control in pharmaceutical and diagnostic laboratories.

Study Objectives and Overview

This work describes the development and validation of an addition-only tryptic digestion protocol and multiplexed SISCAPA peptide enrichment automated in 96-well format on the Agilent Bravo platform. The complete workflow—from plasma denaturation to LC-TQMS quantitation—requires as little as 4 hours for sample preparation and achieves high throughput with a 3 minute LC/MS cycle per sample.

Methodology and Instrumentation

The protocol comprises:

• Addition-only digestion: dried denaturation mix, alkylation, dilution, and trypsin (1:10 enzyme:protein) in 10 µL plasma, incubated at 37 °C.
• Quenching and stable isotope-labeled standard (SIS) peptide spiking.
• SISCAPA enrichment: magnetic anti-peptide antibodies capture target peptides, followed by three washes using a custom SISCAPA magnet array and acid elution.
• LC-TQMS analysis: Agilent 1290 Infinity UHPLC and 6490 triple quadrupole MS; data acquired and processed with Agilent MassHunter software.

Main Results and Discussion

• Sensitivity and Linearity: Limits of detection ranged from sub-femtomole to low-femtomole per 10 µL plasma, with limits of quantitation at similar levels. Calibration curves for four target peptides exhibited R2 > 0.99 and reliable recovery across a 0–150 µL plasma range.
• Accuracy: Comparison of soluble transferrin receptor measurements by automated SISCAPA and a Roche/Hitachi clinical analyzer revealed excellent correlation (R2 = 0.998, slope ≈ 0.196, minimal intercept).
• Precision: Workflow coefficients of variation remained below 7 % (n = 36 samples over three days); digestion-only contribution to variability was under 2.2 %.
• Throughput: Automated preparation of 96 samples in parallel within 4 hours, with a subsequent 3 minute LC/MS cycle time per sample. Adaptation to RapidFire MS can reduce cycle time to ~10 seconds per sample.

Benefits and Practical Applications

• High reproducibility and low variability support routine biomarker quantitation in clinical research and diagnostic laboratories.
• Multiplex capability enables simultaneous measurement of multiple protein targets in minimal sample volumes.
• Scalable format and rapid cycle times facilitate large-scale studies, quality assurance, and industrial workflows.

Future Trends and Opportunities

Continued integration of ultra-high-throughput MS platforms (e.g., RapidFire), expanded antibody panels for broader proteome coverage, and coupling with automated data analytics and AI-driven decision support will further enhance sensitivity, speed, and applicability in precision medicine and biomanufacturing.

Conclusion

The automated addition-only digestion and SISCAPA-MS workflow on the Agilent Bravo platform achieves rapid, sensitive, and highly reproducible quantitation of plasma proteins. Its scalability and throughput render it well suited for clinical biomarker verification, industrial analytics, and large-scale proteomic investigations.

Reference

1. Rittenberg D, Foster GL. A new procedure for quantitative analysis by isotope dilution with application to the determination of amino acids and fatty acids. J Biol Chem. 1940;133:737-744.
2. Anderson L, et al. Mass spectrometric quantitation of peptides and proteins using stable isotope standards and capture by anti-peptide antibodies. J Proteome Res. 2004;3:235-244.
3. Anderson L, et al. High-throughput SISCAPA-based peptide quantitation using an Agilent RapidFire high-throughput MS system. Agilent Application Note 5991-0380EN. 2014.