A Semi Quantitative Method for the Analysis of Tryptic Peptides in Human Serum: A Rapid, Targeted UPLC-MS/MS Approach Using Biognosys Plasma Dive Kit

Significance of the Topic

A rapid, targeted assay for tryptic peptides in human serum addresses a critical need for high-throughput proteomic analysis in research and clinical settings. By leveraging surrogate peptides, large proteins can be monitored quantitatively, facilitating biomarker discovery, therapeutic monitoring, and quality control in large sample cohorts.

Study Objectives and Overview

This work aims to develop and demonstrate a semi-quantitative UPLC-MS/MS workflow capable of analyzing 100 tryptic peptides derived from endogenous serum proteins within a generic LC-MS platform. The approach emphasizes throughput, reproducibility, and versatility across multiple protein targets.

Methodology and Instrumentation

The protocol begins with human serum digestion using the Biognosys PlasmaDive kit: denaturation, reduction, alkylation, tryptic digestion, and spiking with stable isotope-labeled peptide standards. A 6 µL aliquot is injected onto an ACQUITY UPLC I-Class System with a CORTECS T3 2.7 µm, 2.1 × 30 mm column at 60 °C. Peptides are separated over a 2.9-minute gradient from 1 % to 45 % organic, followed by column wash and re-equilibration. MS detection employs a Xevo TQ-S micro triple quadrupole in positive ESI mode, monitoring three MRM transitions per peptide with source parameters fixed (ion source 150 °C, desolvation 650 °C, capillary voltage 2.0 kV). Quantification methods are managed via MassLynx/Quanpedia and Skyline for method creation and data review.

Main Results and Discussion

One hundred serum proteins were represented by unique surrogate peptides and monitored via doubly charged precursors (with two exceptions requiring triply charged precursors). Each peptide was quantified using three b/y fragment ions to ensure confident identification. The 12-minute method accommodates 50 peptides per injection (native and heavy forms), with potential to increase multiplexing by reducing transition counts. Example chromatograms for six representative proteins confirmed robust separation and clear native versus labeled peak alignment.

Benefits and Practical Applications

• High throughput: up to 100 peptides in two runs, suitable for large cohorts.
• Semi-quantitative capability: stable isotope standards enable relative quantification.
• Platform versatility: generic LC-MS setup supports multi-omic workflows (proteomics, metabolomics, lipidomics).
• Scalability: transition management allows customizable multiplexing.

Future Trends and Opportunities

Advances may include further multiplex expansion by optimizing transition selection, integration with automated sample prep, and coupling with emerging data analysis platforms powered by machine learning. The workflow can evolve toward clinical validation, real-time plasma proteomics, and combined multi-omic panels for precision medicine applications.

Conclusion

This targeted semi-quantitative UPLC-MS/MS method delivers a rapid, robust, and versatile approach to serum protein profiling. By employing a generic LC-MS platform and stable isotope standards, it addresses the demands of high-throughput research and lays the foundation for comprehensive multi-omic analyses.

References

1. Billy Joe Molloy. "A Semi Quantitative Method for the Analysis of Tryptic Peptides in Human Serum: A Rapid, Targeted UPLC-MS/MS Approach Using Biognosys Plasma Dive Kit." Waters Corporation, 2018.