Rapid assay development for low input targeted proteomics using a versatile linear ion trap.

Area of Science:

• Biochemistry
• Analytical Chemistry
• Mass Spectrometry

Background:

• High-mass accuracy instruments are typically needed for proteomics on limited cell populations.
• Triple quadrupoles are limited to targeted proteomics, while linear ion traps (LITs) offer versatility.
• There is a need for cost-effective, accessible mass spectrometry solutions in research labs.

Purpose of the Study:

• To describe a workflow for developing targeted proteomics assays from global data-independent acquisition (DIA) measurements using a hybrid quadrupole-LIT instrument.
• To demonstrate the capability of this workflow for quantifying low-level proteins without high-mass accuracy.
• To validate the method's performance in complex biological samples.

Main Methods:

• Utilized a hybrid quadrupole-LIT mass spectrometry instrument.
• Developed an automated software approach for scheduling parallel reaction monitoring (PRM) assays.
• Performed global data-independent acquisition (DIA) to generate assay parameters.
• Quantified low-abundance proteins in a 1 ng background proteome.

Main Results:

• Achieved consistent quantification across three orders of magnitude in a matched-matrix background.
• Demonstrated quantitative linearity for low-level proteins like transcription factors and cytokines below two orders of magnitude.
• Showed consistency between flow cytometry and LIT-based proteomics for T cell subsets from a 1 ng sample.
• Validated the method without the need for stable isotope-labeled standards.

Conclusions:

• Hybrid quadrupole-LIT instruments offer a valuable and accessible solution for targeted proteomics.
• The described workflow enables rapid assay development and sensitive protein quantification from limited samples.
• This approach expands the accessibility of mass spectrometry for diverse laboratory settings.