Intra-sample three-point quantitative calibration using metabolically labelled QconCATs

Quantitative concatamers (QconCATs) have been used since almost 20 years as internal quantification standards for multi-protein panels in targeted and untargeted proteomics assays. Classical approaches use heavy stable isotope labelled QconCATs for single-point internal calibration. The most common labelling strategy uses the labelled amino acids arginine and lysine (SILAC).

Here, we show the advantages and drawbacks of metabolic QconCAT labelling using 15N salt and 13C glucose compared to classical SILAC labelling. By spiking multiple differently labelled QconCATs at increasing concentrations into the same sample, we establish an intra-sample three-point calibration curve. Next, we compare the quantitative performance of intra-sample calibration in a multi-protein MRM panel with one-point calibration and inter-sample multi-point calibration.