Development of Highly Multiplex Targeted Proteomics Assays in Biofluids Using a Nominal Mass Ion Trap Mass...

Area of Science:

• Biochemistry and Proteomics
• Biomedical Engineering
• Clinical Diagnostics

Background:

• Targeted protein assays are vital for clinical studies but struggle with scalability.
• Current methods limit the number of proteins that can be monitored simultaneously.
• Bridging the gap between discovery and clinical application requires scalable assay development.

Purpose of the Study:

• To demonstrate the generation of large-scale targeted protein assays using a novel hybrid nominal mass instrument.
• To showcase the Stellar mass spectrometer's capability for high-throughput protein quantification.
• To validate the workflow in clinical applications, including neurodegenerative disease and extracellular vesicle analysis.

Main Methods:

• Utilized a novel hybrid nominal mass instrument (Stellar mass spectrometer) for assay generation.
• Employed gas-phase fractionated (GPF) data-independent acquisition (DIA) for assay construction.
• Implemented real-time alignment to manage shifting retention times for scalability.
• Compared quantification using Orbitrap DIA and linear ion trap parallel reaction monitoring (PRM).

Main Results:

• Successfully generated large-scale targeted protein assays with high sensitivity and speed.
• Demonstrated the ability to schedule methods from diverse GPF DIA libraries (Orbitrap and linear ion trap).
• Applied the workflow to cerebrospinal fluid (CSF) for Alzheimer's disease-associated proteins and plasma extracellular vesicles (EVs), observing disease-specific differences.

Conclusions:

• The Stellar mass spectrometer enables scalable, sensitive, and fast targeted protein assays for clinical research.
• The developed workflow effectively monitors disease-associated proteins in CSF and EVs.
• This technology facilitates the transition of protein biomarker discovery into large-scale clinical studies.