Subcellular fractionation of human liver reveals limits in global proteomic quantification from isolated fractions.
The liver plays an important role in metabolism and elimination of xenobiotics, including drugs. Determination of concentrations of proteins involved in uptake, distribution, metabolism, and excretion of xenobiotics is required to understand and predict elimination mechanisms in this tissue. In this work, we have fractionated homogenates of snap-frozen human liver by differential centrifugation and performed quantitative mass spectrometry-based proteomic analysis of each fraction. Concentrations of proteins were calculated by the "total protein approach". A total of 4586 proteins were identified by at least five peptides and were quantified in all fractions. We found that the xenobiotics transporters of the canalicular and basolateral membranes were differentially enriched in the subcellular fractions and that phase I and II metabolizing enzymes, the cytochrome P450s and the UDP-glucuronyl transferases, have complex subcellular distributions. These findings show that there is no simple way to scale the data from measurements in arbitrarily selected membrane fractions using a single scaling factor for all the proteins of interest. This study also provides the first absolute quantitative subcellular catalog of human liver proteins obtained from frozen tissue specimens. Our data provide quantitative insights into the subcellular distribution of proteins and can be used as a guide for development of fractionation procedures.
Biochemical Proteomics Group, Department of Proteomics and Signal Transduction, Max Planck Institute of Biochemistry, D-82152 Martinsried, Germany. Electronic address: jwisniew@biochem.mpg.de.
Jacek R
JR
Wiśniewski
Department of Pharmacy, Uppsala University, S-751 23 Uppsala, Sweden; Cardiovascular and Metabolic Diseases, Innovative Medicines and Early Development Biotech Unit, AstraZeneca, Pepparedsleden 1, Mölndal, S-431 83, Sweden.
Christine
C
Wegler
Department of Pharmacy, Uppsala University, S-751 23 Uppsala, Sweden; Science for Life Laboratory, Uppsala University, S-751 23 Uppsala, Sweden.
Per
P
Artursson
D002458:Cell Fractionation / Q000379:methods*; D005260:Female; D006801:Humans; D008099:Liver / Q000737:chemistry*; D008297:Male; D020543:Proteome / Q000737:chemistry*; D040901:Proteomics / Q000379:methods*
D020543:Proteome
Absolute quantitative proteomics; Drug metabolism; Drug transport; Human liver; Subcellular fractionation; Total protein approach