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

Absolute quantification of proteins by mass spectrometry (MS)-based methods is useful in analytical medicine and systems biology. To push the boundaries of protein quantification performance, we developed the PSAQ⁺¹ method. PSAQ⁺¹ uses a protein standard that is minimally distinct from the analyte, a recombinant analogue of the protein target, labeled with ¹³C₁ on arginine and lysine residues, inducing a single unit mass difference for tryptic peptides. It can therefore be introduced early during the analytical workflow to ensure consistent behavior throughout biochemical preparation, liquid chromatography and MS analysis. In this article, we present a proof of concept with an optimized parallel reaction monitoring (PRM) analytical workflow targeting a single composite isotopic peak for each pair of signature peptides. To demultiplex signals originating from the coanalysis of the PSAQ⁺¹ and the endogenous peptides, we developed a dedicated precise quantification model, made available as an R-package (rPSAQ). Our results demonstrate that coanalysis allows robust, reproducible, accurate, and specific quantification. Demultiplexing of the MS/MS signal and the accuracy of quantification were validated using the dedicated mathematical model. Compared to conventional isotope dilution approaches, in which the endogenous and labeled peptides are isolated and fragmented independently, PSAQ⁺¹ enables coisolation and cofragmentation of both peptides as well as codetection of fragments. This halves the cycle time required per peptide target, while coisolation in a narrow mass window reduces matrix interferences, improving specificity and sensitivity. The PSAQ⁺¹ method thus represents a promising strategy for accurate protein quantification in highly complex biological matrices.