Lost in Centrifugation. Accounting for Losses in Quantitative Targetted Absolute Proteomics

Lost in Centrifugation! Accounting for Transporter Protein Losses in Quantitative Targeted Absolute Proteomics (QTAP)Matthew D Harwood†‡, Matthew R Russell§, Sibylle Neuhoff‡, Geoffrey Warhurst†, Amin Rostami-Hodjegan1Gut Barrier Group, Inflammation & Repair, University of Manchester, Salford Royal NHS Trust, Salford, M6 8HD, UK2Simcyp Limited (a Certara Company), Blades Enterprise Centre, Sheffield, S2 4SU, UK3Centre for Applied Pharmacokinetic Research, , Manchester Pharmacy School, Stopford Building, Oxford Road, Manchester, UK, M13 9PT, UKIn drug development, considerable efforts are made to extrapolate from in vitro and pre-clinical findings to predict human drug disposition using in vitro - in vivo extrapolation (IVIVE) approaches. Employing IVIVE strategies linked with Physiologically-Based Pharmacokinetic (PBPK) modeling is widespread and regulatory agencies are accepting and occasionally requesting model analysis to support licensing submissions. Recently, there has been a drive to improve PBPK models by characterizing the absolute abundance of enzymes, transporters and receptors within mammalian tissues and in vitro experimental systems using Quantitative Targeted Absolute Proteomics (QTAP). The absolute abundance of functional proteins relevant to processes governing drug disposition provided by QTAP will enable IVIVE-PBPK to incorporate terms for the abundance of enzymes and transporters in target populations. However, the majority of studies that report absolute abundances of enzymes and transporter proteins do so in enriched membrane fractions, to increase the abundance per sample and thus the assay’s sensitivity, rather than measuring the expected lower abundance in the more biologically meaningful whole cells or tissues. This work will discuss the balance between protein enrichment and loss during the preparation of membrane fractions from whole cells or tissues. Accounting for losses, with recovery factors, throughout the fractionation procedure, provides a means to correct for procedural losses enabling the scaling of protein abundance from subcellular fractions to whole cell or organ abundances. PBPK models based on corrected abundances will more closely resemble biological systems, thereby facilitating the development of more meaningful IVIVE scaling factors producing more accurate quantitative predictions of drug disposition.