Review
Quantitative Targeted Absolute Proteomics-Based Adme Research as A New Path to Drug Discovery and Development: Methodology, Advantages, Strategy, and Prospects

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Abstract

An understanding of the functional roles of proteins, for example, in drug absorption, distribution, metabolism, elimination, toxicity, and efficacy (ADMET/efficacy), is important for drug discovery and development. Equally, detailed information about protein expression is required. Recently, a new protein quantification method, called quantitative targeted absolute proteomics (QTAP), has been developed on the basis of separation and identification of protein digests by liquid chromatography–linked tandem mass spectrometry with multiple reaction monitoring. Target peptides for quantification are selected only from sequence information, so time-consuming procedures such as antibody preparation and protein purification are unnecessary. In this review, we introduce the technical features of QTAP and summarize its advantages with reference to recently reported results. These include the evaluation of species differences of blood–brain barrier protein levels among human, monkey, and mouse. The high selectivity of QTAP and its ability to quantify multiple proteins simultaneously make it possible to determine the absolute expression levels of many proteins in tissues and cells in both physiological and disease states. Knowledge of absolute expression amounts, together with data on intrinsic protein activity, allows us to reconstruct in vivo protein function, and this should be an efficient strategy to predict ADMET/efficacy of drug candidates in humans in various disease states. © 2011 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 100:3547–3559, 2011

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INTRODCUTION

Transporters, enzymes, receptors, and channels are well known to play key roles in drug absorption, distribution, metabolism, elimination, and toxicity (ADMET) and therefore have an important influence on drug efficacy. Both pharmacogenomics and pharmacogenetics approaches have been used in drug discovery and development to elucidate the functions of these proteins in ADMET research,1,2 as well as to establish the extent of interindividual variability. Recent advances in mass spectrometry (MS)

Protein Quantification by MRM Using LC–MS/MS

Quantification by LC–MS/MS has been performed for small molecular compounds, such as drugs, and it was considered that a similar quantification strategy might be available for proteins. However, proteins are too large to separate by reversed-phase high-performance liquid chromatography (HPLC) and their mass lies above the range of the mass filter in MS for quantification [mass-to-charge ratio (m/z) <2800 in 4000QTRAP, <1250 in API5000 (ABSCIEX, Foster City, CA)]. Therefore, proteins must first

Sequence-based Target Peptide Selection for Multiplexed-MRM Analysis

The most important issue for high sensitivity and reliability in protein quantification by MRM analysis is the selection of the target peptide. The target peptide should have a unique amino acid sequence and give a high intensity in MS analysis. One selection strategy has been to identify the target peptide by global proteomics. Picotti et al.5 have developed a database of peptides based on previous proteomics experiments, called the MRM Atlas, and they reported an analysis of 1500 yeast

Comparison of LC–MS/MS-Based Quantification with Antibody-based Quantification

The principle of protein quantification using LC–MS/MS is to quantify specific peptides generated from target proteins by trypsin digestion. As MS selects the peptide based on molecular weight (m/z) with a mass filter, a major advantage of this quantification method is high selectivity, in contrast to the issue of cross-reactivity in antibody-based methods. A single amino acid difference in a peptide changes the m/z, and is able to be distinguished by MS (Table 1). An example of the superiority

Quantification of Membrane Transporter Protein

Membrane proteins are difficult to analyze quantitatively by western blotting or ELISA due to their poor solubility and high aggregability, although membrane proteins include important drug targets and play important roles in ADMET. By applying multiplexed-MRM analysis, we have quantified 43 transporters and Na+/K+ ATPase in mouse; 47 transporters, three junction proteins, two receptors, and Na+/K+ ATPase in monkey; and 106 transporters, 14 membrane proteins, and Na+/K+ ATPase in human

Reconstruction of in Vivo Transporter Protein Function

Variability, such as species difference, in vitro/in vivo difference, age difference, and normal/disease difference, in physiological function must be taken into account in drug discovery and development, as well as clinical application. Drug concentrations in the tissue and plasma, pharmacological and/or toxicological responses, and the pathophysiological state of the drug target organ depend upon the activities of functional proteins such as transporters, enzymes, channels, receptors, and so

QTAP-based Drug Discovery and Development

Great efforts have been directed to drug discovery and development in both the pharmaceutical industry and academia in the past decades. There have been remarkable developments in the methodology of life science, including whole-genome sequencing, genome-wide association study, and global proteomics, which have facilitated the discovery of disease-related proteins, but we are still far away from a “gold-rush era” of new drugs for these target proteins. The processes of drug ADMET/efficacy are

CONFLICT OF INTEREST

Tetsuya Terasaki and Sumio Ohtsuki are a full professor and an associate professor of Tohoku University (Sendai, Japan), respectively, and are also directors of Proteomedix Frontiers. This research was not supported by Proteomedix Frontiers and their positions at Proteomedix Frontiers do not present any financial conflicts. The other authors declared no conflict of interest.

Acknowledgements

The studies mentioned in this review were supported in part by a Grant for Development of Creative Technology Seeds Supporting Program for Creating University Ventures from Japan Science and Technology Agency (JST), and the Industrial Technology Research Grant Program from New Energy and the Industrial Technology Development Organization of Japan, as well as a Grant-in-Aid for Scientific Research (S) 18109002 from the JSPS, a Grant-in-Aid for Scientific Research on Priority Area 17081002 from

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