Rapid Qualitative Analysis and Absolute Quantification of Plasma Proteins Using SONAR with Biognosys PQ500 for Proteomic Clinical Research Studies

Significance of the Topic

Quantitative proteomics plays a pivotal role in clinical research by enabling precise measurement of protein concentrations in complex biological samples. Absolute quantification of plasma proteins supports biomarker discovery, disease monitoring, and therapeutic evaluation. High-throughput approaches that combine specificity, sensitivity, and speed are essential to process large patient cohorts and deliver reproducible data for translational studies.

Study Objectives and Overview

This application note evaluates the performance of the SONAR data‐independent acquisition (DIA) workflow in combination with the Biognosys PQ500 peptide kit for rapid qualitative profiling and absolute quantification of plasma proteins. Human plasma samples from healthy controls, COPD patients, and asthma patients were analyzed to demonstrate identification depth, quantitation precision, and the ability to discriminate clinical cohorts under both 45-minute and 15-minute chromatographic gradients.

Methodology

Sample Preparation:

• Undepleted human plasma from controls (n=6), COPD (n=6), and asthma (n=6).
• Reduction, alkylation, and overnight tryptic digestion.
• Pooled by cohort and spiked with PQ500 stable isotope-labeled peptide standards.

Chromatography and Mass Spectrometry:

• Reversed-phase separation on a peptide CSH C18 column at 55 °C with a 3–40% acetonitrile gradient (0.1% formic acid) over 15 or 45 minutes.
• Flow rate of 7 µL/min, injection of 5 µL digest (5 µg peptide).
• Data acquisition by SONAR DIA with alternating low and elevated collision energy scans, quadrupole linear scanning (330–1100 m/z), and TOF detection (50–2000 m/z).

Used Instrumentation

• ACQUITY UPLC M-Class System with Peptide CSH C18 column
• Xevo G2-XS QTof mass spectrometer operated in ESI(+) mode
• Data processing with Spectronaut Pulsar X and Progenesis QI; statistical analysis via MetaboAnalyst

Main Results and Discussion

The SONAR approach yielded 347 protein group identifications with the 45-minute gradient and 218 with the 15-minute gradient. Principal component analysis and hierarchical clustering clearly separated control, COPD, and asthma cohorts under both conditions. The fast-scanning quadrupole mechanism minimized interference from co-eluting peptides, enabling extraction of clean MS2 fragment ion signals over a dynamic range exceeding four orders of magnitude. Quantitative precision remained robust, with average coefficients of variation (CV) of 6% across technical replicates for both gradient lengths, and all measurements exhibiting CVs below 20%. Absolute concentrations were determined for PQ500-targeted proteins and extended to additional identified plasma proteins beyond the kit’s panel.

Method Benefits and Practical Applications

• High throughput capability supports large clinical datasets with gradient times down to 15 minutes.
• Absolute quantification using stable Isotope-labeled standards yields reproducible protein concentration measurements.
• Enhanced selectivity from scanning quadrupole DIA reduces interference and improves data quality.
• Wide dynamic range detection facilitates quantitation of both high- and low-abundance plasma proteins.
• Applicable to biomarker discovery, disease stratification, and pharmacodynamic studies.

Future Trends and Potential Applications

Emerging developments may include expansion of peptide panels to cover thousands of targets, integration of faster chromatography methods, and incorporation of machine learning for automated data interpretation. The SONAR workflow can be adapted to other biofluids or tissue digests, supporting broader translational proteomics efforts and real-time clinical decision-making.

Conclusion

The coupled SONAR DIA and PQ500 kit workflow delivers rapid, reproducible, and absolute quantification of plasma proteins, demonstrating consistent performance across different gradient times and clinical cohorts. Its high specificity, quantitative precision, and throughput make it an attractive solution for proteomic clinical research and biomarker validation.

References

1. Chong J, et al. MetaboAnalyst 4.0: Towards More Transparent and Integrative Metabolomics Analysis. Nucleic Acids Res. doi:10.1093/nar/gky310.
2. Gethings LA, et al. Lipid Profiling of Complex Biological Mixtures by Liquid Chromatography/Mass Spectrometry Using a Novel Scanning Quadrupole Data-Independent Acquisition Strategy. Rapid Commun Mass Spectrom. 2017;31(19):1599–1606. doi:10.1002/rcm.7941.
3. Moseley AM, et al. Scanning Quadrupole Data-Independent Acquisition, Part A: Qualitative and Quantitative Characterization. J Proteome Res. 2018;17(2):770–779. doi:10.1021/acs.jproteome.7b00464.