A4 AN LC-MS/MS PROTEOMICS BASED METHODOLOGY TO SIMULTANEOUSLY EVALUATE THE RECOVERY, ENRICHMENT AND PURITY OF MICROSOMAL AND CYTOSOLIC FRACTIONS ISOLATED FROM TISSUES FOR IN VITRO DRUG METABOLISM STUDIES

Meijuan Xu , Affiliated Hospital of Nanjing University of Chinese Medicine, Nanjing, China
Marc Vrana , University of Washington, Seattle, WA
Haeyoung Zhang , University of Washington, Seattle, WA
Bhagwat Prasad , University of Washington, Seattle, WA
Purpose: In vitro drug metabolism data obtained in the enriched subcellular fractions such as microsomes and cytosol derived by differential centrifugation are commonly utilized for the in vitro to in vivo extrapolation (IVIVE) of drug clearance. The purity of subcellular fractions and the accurate estimation of physiologically relevant scaling factor, i.e., microsomal or cytosolic protein per gram of tissue, are the key parameters in the implementation of IVIVE. Therefore, the specific aims of this study were: 1) to develop an LC-MS/MS proteomics method for simultaneous quantification of various subcellular organelle markers, and 2) to apply this method in determining the recovery, enrichment, and purity of microsomal and cytosolic fractions isolated from human liver tissue.

Methods: One to three protein markers of microsomes, cytosol, plasma membrane, mitochondria, nucleus, peroxisome, lysosome and cytoskeleton, which are ubiquitously expressed across human tissues, were selected. Various in silico and experimental approaches were used to select surrogate peptides and optimize LC-MS/MS parameters for quantification of these markers [1]. The protein quantification method was refined and validated by analysis of synthetic heavy labeled peptides. The cytosolic and microsomal fractions were isolated from liver tissues from three donors using differential centrifugation procedure. 16 protein markers were quantified in these subcellular fractions after trypsin digestion, and their recovery, enrichment and purity were calculated.

Results: The robustness of the relative protein quantification was confirmed by good correlations between different MRM transitions (r>0.93) for each peptide and between surrogate peptides (r>0.92) for each marker. The recovery of the two endoplasmic reticulum (ER) membrane markers (calnexin and NADPH cytochrome P450 reductase) were consistent across samples (37.6±10.7% and 36.2±7.5%). The recovery of ER lumen marker (calreticulin) in the microsomal fractions was significantly lower (6.9±0.9%) than the cytosolic fraction (55.7±11.2%). High recovery (68.0-78.3%) of cytosolic makers (Hsp90 and lactate dehydrogenase) was observed in the cytosolic fraction. The enrichments of the ER membrane and cytosolic markers in the microsomal and cytosolic fractions were around 5- and 2- fold, respectively. The cytosolic fraction showed significant contamination of cytoskeleton, ER lumen, peroxisome matrix, mitochondria matrix, lysosome lumen and nucleus membrane markers, whereas, impurity of lysosomal and peroxisomal membrane was detected in the microsomal fraction.

Conclusions: An LC-MS/MS method was successfully developed and applied to determine the recovery, enrichment and contamination of microsomal and cytosolic fractions of the human liver tissue. Significant loss during the processing and impurity of other organelle markers in microsomes and cytosol fractions necessitate the need of optimum characterization of these in vitro models prior to application in the IVIVE. This approach can be used for optimization and batch-to-batch quality control of the subcellular isolation methodology. The methodology also has application in characterizing subcellular localization of drug metabolizing enzymes and transporters.

1. Vrana, M., et al., Database of Optimized Proteomic Quantitative Methods for Human Drug Disposition-Related Proteins for Applications in Physiologically Based Pharmacokinetic Modeling. CPT Pharmacometrics Syst Pharmacol, 2017. 6(4): p. 267-276.