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Abstract
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).
Detergent-based protein extraction and fractionation of PBMC
Surrogate peptides for selective protein analysis by MS/MS after tryptic digestion
Filter-aided sample preparation (FASP) for whole-proteome analysis
Immunoaffinity enrichment of proteins from PBMC lysates
Representative multiplex analysis from 1mL of whole blood
Multiplex analysis of endogenous CD3, CD4, and CD8
Optimization of tryptic digestion conditions
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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