Targeted proteomic analysis of human plasma on a discovery scale

Significance of the Topic

Human plasma proteins reflect organ physiology and disease states making plasma an attractive source of biomarkers. Conventional immunoassays suffer from limited specificity and multiplexing capacity. Targeted mass spectrometry provides high specificity and the ability to quantify hundreds of proteins simultaneously in a single run.

Aims and Study Overview

This study presents a clinical research workflow combining PQ500 reference peptides with a Thermo TSQ Altis triple quadrupole mass spectrometer and capillary flow LC to enable reliable targeted quantification of more than 500 human plasma proteins in 70 minutes per sample. A cohort of cancer patients and healthy donors was analyzed to demonstrate performance.

Methodology and Instrumentation

Sample preparation used a high throughput kit for plasma digestion and spiking of 804 stable isotope labelled peptides representing 500 proteins. Chromatography was performed on a 15 cm CSH C18 column in direct injection mode with a 60 minute linear gradient at 5 µl/min. Data acquisition employed scheduled SRM/MRM with three transitions per peptide, 2.5 minute retention time windows and a 2.5 second cycle time. SpectroDive software was used for assay scheduling, data extraction and statistical analysis.

Main Results and Discussion

• Retention time calibration based on all SIS peptides achieved a median offset of 25 seconds and optimized 2.5 minute windows allowing 480 concurrent transitions.
• Out of 804 peptides, 756 were reproducibly detected; more than 300 plasma proteins were quantified per individual sample at 1 % FDR.
• Precision assessment in pooled plasma triplicates yielded a median CV of 5.4 % with 271 proteins below 20 % CV.
• Statistical comparison revealed significant elevation of inflammation markers such as C reactive protein and S100 A9 in cancer patients.
• LOD and LOQ assessment using a serial dilution demonstrated median LODs in the atto mole range for 790 peptides, confirming excellent sensitivity and linearity.

Benefits and Practical Application

This method matches the proteome coverage of discovery experiments while delivering superior quantitative precision and throughput of up to 20 samples per day. It is well suited for clinical research applications including biomarker validation, patient stratification and personalized medicine.

Future Trends and Opportunities

Integration with larger clinical cohorts and multi omics workflows, automation of sample preparation and further advances in targeted software could expand the utility of this approach. Emerging high throughput instrumentation may enable deeper coverage and faster turnaround in precision medicine studies.

Conclusion

The combination of PQ500 reference peptides, TSQ Altis mass spectrometer and SpectroDive software provides a robust and sensitive targeted proteomics workflow. It enables reliable quantification of several hundred plasma proteins with high precision in a clinical research context.

Used Instrumentation

• Thermo Scientific TSQ Altis triple quadrupole mass spectrometer
• Thermo Scientific UltiMate 3000 RSLCnano system
• Capillary flow LC CSH C18 column (Waters)
• Biognosys PQ500 stable isotope reference peptide mix
• SpectroDive targeted proteomics software

References

• Baker M. Antibody anarchy A call to order Nature 527 545–551 2015