Isotopologue Multipoint Calibration for Proteomics Biomarker Quantification in Clinical Practice

Area of Science:

• Biochemistry
• Analytical Chemistry
• Biomarker Discovery

Background:

• Targeted proteomics is crucial for biomarker validation in human biopsies.
• Clinical translation requires reduced complexity, simplified procedures, and improved throughput in targeted proteomics.

Purpose of the Study:

• To present the Isotopologue Multipoint Calibration (ImCal) quantification strategy.
• To enable accurate quantification of biomarker peptides in clinical applications without expert supervision.

Main Methods:

• Developed ImCal using five isotopically-labeled peptides at varying concentrations for internal multipoint calibration curves.
• Applied ImCal to quantify HER2 in 35 breast cancer formalin-fixed paraffin-embedded patient samples.

Main Results:

• ImCal provides accurate quantification of endogenous peptides using internal multipoint calibration curves.
• Demonstrated high heterogeneity in HER2 levels among breast cancer patients, contrasting with homogeneous immunohistochemistry classification.
• Validated ImCal's compatibility with various mass spectrometers and its ability to assess matrix effects and quantification ranges.

Conclusions:

• ImCal simplifies targeted proteomics for clinical routine, enhancing analytical performance.
• Improved mass spectrometry methods like ImCal facilitate fine-grain patient stratification for better disease diagnosis and prognosis.