Targeted Protein Quantification Using Parallel Reaction Monitoring (PRM)

Area of Science:

• Biochemistry
• Analytical Chemistry
• Mass Spectrometry

Background:

• Targeted proteomics enables sensitive and accurate protein quantification.
• Parallel Reaction Monitoring (PRM) on hybrid instruments offers high-resolution MS/MS analysis for peptide identification and quantification.
• Stable isotope-labeled peptides are crucial for precision in PRM by correcting technical variations.

Purpose of the Study:

• To describe the development of a robust PRM assay for protein quantification.
• To outline the selection criteria for unique surrogate peptides representing target proteins.
• To introduce the open-access tool CalibraCurve for assay validation.

Main Methods:

• Development of a PRM assay for targeted protein quantification.
• Selection of specific peptides as unique surrogates for target proteins.
• Generation of calibration curves and determination of linear concentration ranges and limits of quantification (LOQ) using CalibraCurve.

Main Results:

• A validated PRM assay for targeted protein quantification.
• Established criteria for selecting optimal surrogate peptides.
• Demonstrated the utility of CalibraCurve for determining assay performance metrics.

Conclusions:

• The developed PRM assay provides a sensitive and accurate method for protein quantification.
• The CalibraCurve tool facilitates the validation of PRM assays by defining concentration ranges and LOQ.
• This work advances targeted proteomics by providing a standardized method development and validation approach.