Isotopologue Multipoint Calibration for Proteomics Biomarker Quantification in Clinical Practice

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 for each
individual sample and to accurately quantify biomarker peptides in
clinical applications without the need of expert supervision. ImCal
relies on the use of five different isotopically-labelled peptides
of different nominal mass mixed at different concentrations to be
used as an internal calibration curve for each endogenous peptide.
The use of internal multipoint calibration curves is well-suited for
the generation of ready-to-use biomarker kits for clinical applications
as it is compatible with both high- and low-resolution mass spectrometers
and different levels of endogenous peptide, it eliminates the need
for blank matrixes required in external curves, it allows the evaluation
of matrix effects and the valid quantification range in each individual
sample, and it does not require expert adjustment. We used the ImCal
method to quantify HER2 in 35 breast cancer formalin-fixed paraffin-embedded
patient samples, revealing a high degree of heterogeneity among patients,
which contrasts with the homogeneous immunohistochemistry patient
classification. Our work illustrates how an improvement of mass spectrometry
methods for biomarker quantification can provide fine-grain patient
stratification, and thus better disease diagnostic and prognosis.