Stable Isotope-Triggered Offset Fragmentation Allows Massively Multiplexed Target Profiling on Quadrupole-Orbitrap...

Area of Science:

• Proteomics
• Mass Spectrometry
• Biochemistry

Background:

• Parallel-reaction monitoring (PRM) is a key technique for targeted protein analysis using high-resolution, accurate-mass (HR/AM) Orbitrap mass spectrometry.
• A major limitation of PRM is its restricted multiplexing capacity, primarily due to reliance on reproducible peptide retention times.

Purpose of the Study:

• To develop a novel data acquisition method for Q Exactive mass spectrometers that facilitates retention-time-independent, massive multiplexing for targeted protein analysis.
• To establish an easily applicable method that overcomes the multiplexing limitations of conventional PRM.

Main Methods:

• Introduced pseudo-parallel reaction monitoring (pseudo-PRM), a data-dependent acquisition strategy.
• Utilized high-intensity stable isotope-labeled peptides to trigger the fragmentation of corresponding light peptides.
• Analyzed pseudo-PRM data using standard PRM analysis workflows.

Main Results:

• Demonstrated good reproducibility and sensitivity comparable to standard PRM across various biological samples (yeast, human cells, serum).
• Validated pseudo-PRM for accurate and precise quantification of target peptides using both precursor and fragment ion areas.
• Achieved multiplexing of over 1000 targets in a single analytical run.
• Successfully applied pseudo-PRM to quantify vaccinia virus proteins during infection.

Conclusions:

• Pseudo-PRM is a viable alternative method for multiplexed target profiling on Q Exactive mass spectrometers.
• The method overcomes PRM's retention time dependency, enabling significantly higher multiplexing capacity.
• Pseudo-PRM provides a robust platform for sensitive and accurate targeted protein quantification.