PSAQ⁺¹: Absolute Protein Quantification Using a ¹³C₁-Labeled Protein Standard, Coisolation of Peptide Pairs and...

Area of Science:

• Proteomics
• Analytical Chemistry
• Systems Biology

Background:

• Mass spectrometry (MS)-based absolute protein quantification is crucial for analytical medicine and systems biology.
• Existing methods face challenges in achieving high performance and efficiency.
• There is a need for improved techniques for accurate protein quantification in complex biological samples.

Purpose of the Study:

• To introduce and validate the PSAQ+1 method for enhanced protein quantification.
• To demonstrate the advantages of PSAQ+1 over conventional isotope dilution approaches.
• To provide a robust and efficient tool for accurate protein measurement.

Main Methods:

• Developed the PSAQ+1 method using a minimally distinct, 13C1-labeled recombinant protein standard.
• Implemented an optimized parallel reaction monitoring (PRM) workflow.
• Created a precise quantification model and R-package (rPSAQ) for signal demultiplexing.
• Enabled coisolation, cofragmentation, and codetection of endogenous and labeled peptides.

Main Results:

• PSAQ+1 allows for robust, reproducible, accurate, and specific protein quantification.
• The method halves the cycle time per peptide target compared to conventional approaches.
• Coisolation in a narrow mass window reduces matrix interferences, enhancing specificity and sensitivity.
• Validation using the rPSAQ mathematical model confirmed signal demultiplexing and quantification accuracy.

Conclusions:

• The PSAQ+1 method offers a promising strategy for accurate protein quantification in complex biological matrices.
• Coanalysis of labeled and endogenous peptides significantly improves efficiency and specificity.
• The developed R-package (rPSAQ) provides essential tools for data analysis.