Development and applications of enhanced mass spectrometric quantification of peptide hormones

The analysis of peptide hormones in the clinical lab is currently performed using ligand-binding assays, which suffer from cross-reactivity and poor interlaboratory repeatability, potentially resulting in misdiagnosis. The aim of this project is to improve the analysis of peptide hormones using mass spectrometric detection. To achieve this a collaboration is set up between the University Medical Center Groningen (UMCG) and Spark Holland B.V.

Peptide hormones are important biomarkers in research and diagnosis of endocrine disorders. Quantitative analysis is performed using various ligand-binding assays or enzymatic digestion LC-MS/MS methods, whose widespread adoption is hampered by time-consuming sample preparation procedures. Mass spectrometric quantification of intact peptide hormones provides a novel application that allows for a more reproducible analysis.

They developed a selective protein precipitation strategy that allows for the direct and simultaneous mass spectrometric quantification of IGF-1 and IGF-2. This method can be readily implemented in routine patient care within clinical mass spectrometric laboratories. The two-step selective protein precipitation strategy can be applied to the quantification of different peptide hormones.

A mass spectrometric quantification method is to be developed for different clinically relevant proteoforms of gastrin. This method will be based on the selective precipitation strategy demonstrated in the analysis of IGFs and/or online SPE technologies developed by Spark Holland B.V.

Furthermore, an overview of derivatisation of peptides is to be provided. This subject is relatively unexplored, but shows exciting possibilities for the enhanced mass spectrometric detection of peptides.

In summary the project will provide:

• An LC-MS/MS method for the mass spectrometric quantification of IGF-1 and IGF-2
• Age-related reference values for IGF-1 and IGF-2
• Insight into the analysis of peptide hormones that exist in blood in lower concentrations (gastrin)

Review on the current status of peptide derivatisation of peptides for mass spectrometric analysis.

Results:

The results show that IGF-1 and IGF-2 can be reliably measured by LC-MS/MS in a routine clinical laboratory . The developed method is very simple and straightforward and is suitable for a routine workflow.

Using the newly developed method, age-dependent reference intervals were established. Approximately 1500 healthy individuals and children were used to establish these new reference intervals. These reference intervals will be the new standard for clinical laboratories in the Netherlands.