Quantification of proteins by flow cytometry: Quantification of human hepatic transporter P-gp and OATP1B1 using flow cytometry and mass spectrometry.
Flow cytometry is a powerful tool for the quantitation of fluorescence and is proven to be able to correlate the fluorescence intensity to the number of protein on cells surface. Mass spectroscopy can also be used to determine the number of proteins per cell. Here we have developed two methods, using flow cytometry and mass spectroscopy to quantify number of transporters in human cells. These two approaches were then used to analyse the same samples so that a direct comparison could be made. Transporters have a major impact on the behaviour of a diverse number of drugs in human systems. While active uptake studies by transmembrane protein transporters using model substrates are routinely undertaken in human cell lines and hepatocytes as part of drug discovery and development, the interpretation of these results is currently limited by the inability to quantify the number of transporters present in the test samples. Here we provide a flow cytometric method for accurate quantification of transporter levels both on the cell surface and within the cell, and compare this to a quantitative mass spectrometric approach. Two transporters were selected for the study: OATP1B1 (also known as SLCO1B1, LST-1, OATP-C, OATP2) due to its important role in hepatic drug uptake and elimination; P-gp (also known as P-glycoprotein, MDR1, ABCB1) as a well characterised system and due to its potential impact on oral bioavailability, biliary and renal clearance, and brain penetration of drugs that are substrates for this transporter. In all cases the mass spectrometric method gave higher levels than the flow cytometry method. However, the two methods showed very similar trends in the relative ratios of both transporters in the hepatocyte samples investigated. The P-gp antibody allowed quantitative discrimination between externally facing transporters located in the cytoplasmic membrane and the total number of transporters on and in the cell. The proportion of externally facing transporter varied considerably in the four hepatocyte samples analysed, ranging from only 6% to 35% of intact and viable cells. The sample with only 6% externally facing transporter was further analysed by confocal microscopy which qualitatively confirmed the low level of transporter in the membrane and the large internal population. Here we prove that flow cytometry is an important tool for future protein analysis as it can not only quantify the number of proteins that a cell express but also identify the number of proteins on the surface and it is easy to apply for routine assays.
Bioscience Technology Facility, Department of Biology, University of York, York YO10 5DD, UK.
Karen
K
Hogg
Bioscience Technology Facility, Department of Biology, University of York, York YO10 5DD, UK.
Jerry
J
Thomas
Bioscience Technology Facility, Department of Biology, University of York, York YO10 5DD, UK.
David
D
Ashford
Bioscience Technology Facility, Department of Biology, University of York, York YO10 5DD, UK.
Jared
J
Cartwright
AstraZeneca R&D Alderley Park, Alderley Park, Cheshire, SK10 4TG, UK.
Ruth
R
Coldwell
AstraZeneca R&D Alderley Park, Alderley Park, Cheshire, SK10 4TG, UK.
Daniel J
DJ
Weston
Bioscience Technology Facility, Department of Biology, University of York, York YO10 5DD, UK.
John
J
Pillmoor
AstraZeneca R&D Alderley Park, Alderley Park, Cheshire, SK10 4TG, UK.
Dominic
D
Surry
Bioscience Technology Facility, Department of Biology, University of York, York YO10 5DD, UK. Electronic address: peter.otoole@york.ac.uk.
Peter
P
O'Toole
D018435:ATP Binding Cassette Transporter, Subfamily B / Q000032:analysis; D002460:Cell Line; D005434:Flow Cytometry; D022781:Hepatocytes / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D027381:Liver-Specific Organic Anion Transporter 1; D013058:Mass Spectrometry; D027361:Organic Anion Transporters / Q000032:analysis*
C513055:ABCB1 protein, human; D018435:ATP Binding Cassette Transporter, Subfamily B; D027381:Liver-Specific Organic Anion Transporter 1; D027361:Organic Anion Transporters; C503999:SLCO1B1 protein, human
Flow cytometry; Mass spectrometry; Quantification