Background Quantifying pharmacodynamic biomarker changes enables decision making and clinical trials in drug development. Pharmacodynamic biomarkers are used to determine the effects of treatment on disease. Mass spectrometry offers a quantitative, selective, and multiplex platform for pharmacodynamic protein biomarker analysis in clinical samples (e.g. blood and tumor) that is feasible across multiple sample conditions (e.g. fresh, frozen and formalin-fixed paraffin-embedded (FFPE)). To date, however, methodologies for targeted protein analysis by mass spectrometry (i.e. quantitative proteomics) are underdeveloped for application in immuno-oncology.
Methods To address this, we sought to extract the immuno-oncology-associated T cell membrane proteins CD3, CD4 and CD8 from peripheral blood mononucleate cells (PBMC) and develop a multiplexed mass spectrometry method to quantify their expression. PBMC were isolated from whole blood and using detergent-based lysis buffers fractionated into a cytosolic and membrane protein lysate (figure 1). Analytical methods were then developed to detect proteotypic peptides of all three proteins (table 1 and figure 6) from the lysates by mass spectrometry.
Results CD3, CD4 and CD8 were detected in the membrane protein fraction but not in the cytosolic protein fraction after whole-proteome tryptic digestion using a filter-aided sample preparation (or FASP) technique but with a signal-to-noise ratio of ≤ 2.0 (figure 2). Applying an additional immunoaffinity (IA) enrichment step with antibody-conjugated magnetic beads, prior to digestion, dramatically improved the analyte signal-to-noise ratios to > 100 (figure 3). Reverse-phase nanoflow liquid chromatography (LC) was used to separate all three analytes in multiplex over a 12-minute run prior to tandem mass analysis (MS/MS) (figure 4). Together, this IA-LC-MS/MS method resulted in detection of endogenous CD3, CD4 and CD8 proteins from small volumes of whole blood (< 0.1 mL) and the analyte responses were linear over at least two orders of magnitude (figure 5).
Conclusions This method was developed specifically to quantitate pharmacodynamic changes in CD4 and CD8 T cell membrane expressions from clinically feasible samples (i.e. PBMC). This work, however, provides a foundation for developing methodologies to conduct quantitative proteomics applicable to immuno-oncology, which may be used to interrogate additional pharmacodynamic biomarkers.
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