Targeted proteomics has become the method of choice for biomarker validation in human biopsies due to its high sensitivity, reproducibility, accuracy, and precision. However, for targeted proteomics to be transferred to clinical routine there is the need to reduce its complexity, make its procedures simpler, increase its throughput, and improve its analytical performance. Here we present the Isotopologue Multipoint Calibration (ImCal) quantification strategy, which uses a mix of isotopologue peptides to generate internal multipoint calibration curves to accurately quantify biomarker peptides in clinical applications without the need of expert supervision.
The performance of ImCal was demonstrated quantifying 2 peptides from HER2 in standard and in clinical samples. Five different isotopically-labeled internal standard were synthesized per each peptide of interest. The peptides were isotopologues, with the same sequence but different nominal mass. All samples were analysed in a triple quadrupole in SRM mode.
HER2 protein was quantified from FFPE tissue samples from breast cancer tumours. The isotopologue standard peptides were spiked in the samples at different amounts to generate an internal calibration curve per each endogenous peptide covering 2 orders of magnitude. To determine the accuracy and precision of the internal calibration method in peptide quantification, the method was compared with the standard methods using known amounts of the unlabelled peptide. Results show that ImCal renders high accuracy and reproducibility in peptide quantification, and it is suitable to generate ready-to-use quantification kits for selected biomarkers of clinical relevance.
The use of ImCal overcomes several of the current limitations for peptide biomarker quantification in clinical proteomics applications. It is compatible with both high- and low-resolution mass spectrometers as well as with different levels of endogenous peptide. It eliminates the need for blank matrixes, and it allows the evaluation of matrix effects and definition of a valid quantification range in each individual sample.