Ex vivo glucocorticoid-induced secreted proteome approach for discovery of glucocorticoid-responsive proteins in human serum.

Abstract

To identify glucocorticoid-responsive proteins measurable in human serum that may have clinical utility in therapeutic drug monitoring and the diagnosis of cortisol excess or deficiency. A phased biomarker discovery strategy was conducted in two cohorts. Secretome from peripheral blood mononuclear cells (PBMC) isolated from six volunteers after ex vivo incubation ± dexamethasone (DEX) 100ng/mL for 4 h and 24 h was used for candidate discovery and qualification using untargeted proteomics and a custom multiple reaction monitoring mass spectrometry (MRM-MS) assay, respectively. For validation, five candidates were measured by immunoassay in serum from an independent cohort (n=20), sampled at 1200 h before and after 4mg oral DEX. The discovery secretome proteomics data generated a shortlist of 45 candidates, with 43 measured in the final MRM-MS assay. Differential analysis revealed 16 proteins that were significant in at least one of two time points. In the validation cohort, 3/5 serum proteins were DEX-responsive, two significantly decreased: lysozyme C (p<0.0001) and nucleophosmin-1 (p<0.01), while high mobility group box 2 significantly increased (p<0.01). Using an ex vivo proteomic approach in PBMC, we have identified circulating glucocorticoid-responsive proteins which may have potential as serum biomarkers of glucocorticoid activity.