Quantitative Targeted Absolute Proteomics-Based Adme Research as A New Path to Drug Discovery and Development: Methodology, Advantages, Strategy, and Prospects  Sumio Ohtsuki, Yasuo Uchida, Yoshiyuki Kubo, Tetsuya Terasaki  Journal of Pharmaceutical Sciences  Volume 100, Issue 9, Pages 3547-3559 (September 2011) DOI: 10.1002/jps.22612 Copyright © 2011 Wiley-Liss, Inc. Terms and Conditions

Figure 1 Detection of target peptide by multiple reaction monitoring (MRM) analysis using a triple quadrupole tandem mass spectrometer (Triple Q MS). The total ion chromatogram (TIC) presents the ion intensity of all peptides determined by nanoLC-Orbitrap. The extracted ion chromatogram (XIC) presents ion intensity determined by MRM transition of the target peptide. In MRM analysis, the target peptide is selected by two mass filters (Q1 and Q3), resulted in a highly selective chromatogram, as shown in the right XIC, by reducing noise from the complex peptide sample, shown in the left TIC. Journal of Pharmaceutical Sciences 2011 100, 3547-3559DOI: (10.1002/jps.22612) Copyright © 2011 Wiley-Liss, Inc. Terms and Conditions

Figure 2 Procedure of sample preparation and quantification by MRM analysis. Journal of Pharmaceutical Sciences 2011 100, 3547-3559DOI: (10.1002/jps.22612) Copyright © 2011 Wiley-Liss, Inc. Terms and Conditions

Figure 3 Method development procedures for LC–MS/MS-based protein quantification and quantitative real time RT-PCR. Journal of Pharmaceutical Sciences 2011 100, 3547-3559DOI: (10.1002/jps.22612) Copyright © 2011 Wiley-Liss, Inc. Terms and Conditions

Figure 4 Comparison of absolute protein expression levels of transporters in brain capillaries between (A) human and monkey and (B) human and mouse. The protein expression levels are taken from our previous reports: human,12 monkey,8 and mouse.6 In panel (B), mouse abca8a, abca8b, and abca9 were compared as possible homologues of human ABCA8. Human OATP-A and OATP-B were compared as possible homologues of mouse oatp-2 at the BBB. The solid line passing through the origin represents the line of identity, and the broken lines represent threefold differences. Each point represents mean ± SD. The molecules on the horizontal (mouse and monkey) or vertical (human) axis are under the limits of quantification. Journal of Pharmaceutical Sciences 2011 100, 3547-3559DOI: (10.1002/jps.22612) Copyright © 2011 Wiley-Liss, Inc. Terms and Conditions

Figure 5 Reconstruction of GLUT1 transport activity at in vivo brain capillaries from absolute expression amount and in vitro transport activity. Journal of Pharmaceutical Sciences 2011 100, 3547-3559DOI: (10.1002/jps.22612) Copyright © 2011 Wiley-Liss, Inc. Terms and Conditions

Figure 6 A basic workflow of QTAP-based study. two-dimensional image converted analysis of liquid chromatography and mass spectrometry (2DICAL) is the differential protein expression analysis by label-free quantitative global proteomics developed by Ono et al.46 Journal of Pharmaceutical Sciences 2011 100, 3547-3559DOI: (10.1002/jps.22612) Copyright © 2011 Wiley-Liss, Inc. Terms and Conditions