RUOMKT-02-1903

Quantitative Analysis of Transporter Protein using TripleTOF® 6600 System

For Research Use Only. Not for use in diagnostic procedures

By Xi Chen1, Lei Xiong1, Buyun Chen2, Liling Liu2, Ruina Li2, Suma Ramagiri1, Mollah Sahana1, Emile Plise2, Jonathan Cheong2, Hoa Le2, Yuzhong Deng2, Brian Dean2, Yuan Chen2, Xiaorong Liang2

1SCIEX 1201 Radio Rd, Redwood City, CA 94065; 2Genentech, 1 DNA way, South San Francisco, CA 94080

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Key Challenges in Drug Transporter Study

1. Differential expression – Lack of knowledge of differential transporter protein expression in tissues and cells makes it difficult for in vitro- in vivo correlation (IVIVC).

2. Reliable and reproducible quantitation – Lack of purified standards and hydrophobic properties of membrane proteins presents challenges in a cost effective and reliable qualitative and quantitative analysis.

Figure 1: Showing hepatobiliary drug transporter mechanism; SWATH™ Acquisition and TripleTOF® 6600 system with ProteinPilot™ and MultiQuant™ software.

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Key Benefits of Using TripleTOF® 6600 System for Drug Transporter Assay

1. Qual & Quant workflow in single injection – simultaneous qual and quant using SWATH™ acquisition will help profile differential expression and quantitative analysis in a single experiment.

2. Multicomponent analysis – Ability to retrospectively mine the data and multiplexing capabilities will enable quantitation of multiple transporter proteins without the need for repeated analysis of the same sample.

3. Sensitive and selective assay – High resolution accurate mass quantitation using MRMHR provides with both selective and sensitive LC/MS/MS based assay.

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Unique Features of TripleTOF® 6600 System, SWATH™ Acquisition and MRMHR Assay

1. SWATH™ Acquisition for comprehensive analysis – No method development required to identify and quantitate large number of proteins.

2. MRM like quantitation – High specificity is obtained using MRMHR scan function both in high sensitivity and high resolution mode depending on the need .

3. Fast cycle times maintained – Processing speed on TripleTOF® System allows for sufficient peak coverage, even with fast LC separations without any compromise.

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Introduction

Transport plays an important role in the absorption, distribution, and elimination of a variety of drugs.

In recent years, a large number of transporters, both efflux (ATP-binding cassette (ABC) family) and influx (solute carrier (SLC) family members) have been identified and well characterized in vitro.

However, the abundance of these transporters in the hepatocyte and cell lines as well as in the tissues such as intestine, liver, and kidney has not been accurately quantitated due to technical challenges.

This work aims to build a robust liquid chromatography-mass spectrometry (LC-MS) workflow on the SCIEX TripleTOF® 6600 platform to enable the quantitation of a variety of SLC and ABC drug transporters expressed in the hepatocyte and cell line plasma membranes.

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Materials and Methods

Materials

Peptides and their stable labeled peptides were purchased from New England Peptide, Inc..

Dithiothreitol and ammonium bicarbonate were purchased from Sigma.

Iodoacetamide was obtained from GE Healthcare.

Sequencing grade modified trypsin was purchased from Promega.

MDCK cells transfected with human MDR1 and BCRP or HEK cells transfected with human OATP1B1 and OATP1B3 were grown at Genentech under standard culture conditions.

Human hepatocytes were purchased from Bioreclamation/IVT.

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Materials and Methods

Membrane protein extraction

Native membrane extraction was performed using the manufacturer’s protocol http://www.emdmillipore.com/INTL/en/product/proteoextractnative-membrane-protein-extraction-kit,EMD_BIO- 444810?attachments=TI#anchor_USP

Sample Preparation

Cell membrane extract (2 mg/ml total protein concentration) was denatured, reduced, alkylated, and digested using trypsin overnight. The peptides generated were injected into SCIEX 6600 for analysis.

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LC-MS/MS Conditions

Trypsin digested E. coli BGAL from the SCIEX mass spectrometer standards kit, Part No. 4368624 was diluted to 0.5 pmol/µL in 50% acetonitrile in water with 0.1%formic acid.

Infusion was performed at 2 µL/min using an Eksigent microLC electrode (25 µm) in the Turbo VTM ion source.

LC-MS/MS injections were performed on a 0.10 pmol/µL (5 µL injection) sample at 0.25 mL/min with the standard SCIEX Turbo VTM electrode.

Materials and Methods

LC System Agilent 1290 Infinity HPLC system

Analytical column Phenomenex Kinetex C18 column (3×100 mm, 2.6 μm)

Analytical flow 0.15 ml/min

Mobile Phase A Water (0.1 % formic acid)

Mobile Phase B Acetonitrile (0.1 % formic acid)

Gradient conditions

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MS Conditions

Software

Materials and Methods

MS System SCIEX TripleTOF® 6600 system DuoSpray™ Ion Source

Ionization Mode ESI with Positive Mode Information Dependent Acquisition (IDA) MRMHR and SWATH™ Acquisition

Data acquisition Analyst® TF 1.7 Software

Quantitation ProteinPilot™ Software MultiQuant™ Software

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Results and Discussion

Peptide Sequence Identification by ProteinPilot™ 5.0.

A Paragon algorithm was applied during search in which a generic Uniprot proteome database was used. All 4 target transporters were identified successfully from the human cell line samples. Figure 2 represents the ProteinPilot™ search result for MDR1 membrane protein extraction sample. The presence of MDR1 was confirmed unambiguously with 45 peptides identified. The Fragment Evidence panel solidly supports a good match between the major fragment ions of MS/MS spectrum and the theoretical peptide fragments for each identified peptide. The unique peptides were selected as targets for protein quantitation.

Peptide Quantitation by MRMHR and SWATH™ 2.0 Acquisition

Human cell line samples were quantified using MRMHR and SWATH™ 2.0 acquisition. The eight-point calibration curve was prepared by spiking signature peptides into the ammonium bicarbonate buffer. The concentration of OATP1B1, OATP2B1, MDR1 and BCRP peptides in samples extracted from different cell line were summarized in Table 1.

In MRMHR acquisition, mass transitions 588 (+2) →961, 799 (+2)→712, 468 (+2)→719 and 575 (+2)→664 were utilized, with LLOQs at 0.52, 0.52, 0.26 and 0.52 ng/mL, to monitor OATP1B1, OATP2B1, MDR1, and BCRP peptides, respectively.

In SWATH™ acquisition, a 6 Da acquisition window was applied to cover the mass range between m/z 400-1000. The aforementioned mass transitions were used for quantitation.

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Results and Discussion

Figure 2: Peptide Sequence Identification and Confirmation using ProteinPilot™

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Results and Discussion

Table 1 Peptide Concentration in Cell Line Sample using MRMHR*

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Results and Discussion

Figure 3 Peptide Concentration Comparison in Cell Line Sample (n=3)

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Results and Discussion

Figure 4: Representative Chromatograms of OATP1B1 Peptide acquired in TOF MS mode, MRMHR in high sensitivity mode, SWATH™ acquisition mode on TripleTOF® 6600 System

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Results and Discussion

Figure 4: Representative Chromatograms of OATP1B1 Peptide acquired in TOF MS mode, MRMHR in high sensitivity mode, SWATH™ acquisition mode on TripleTOF® 6600 System

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Results and Discussion

Figure 4: Representative Chromatograms of OATP1B1 Peptide acquired in TOF MS mode, MRMHR in high sensitivity mode, SWATH™ acquisition mode on TripleTOF® 6600 System

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Conclusions and References

Conclusions

1. OATP1B1, OATP2B1, MDR1 and BCRP transporter proteins in the extract of human hepatocyte and different cell lines were successfully identified by IDA and quantified through MRMHR and SWATH™ acquisitions.

2. A comprehensive workflow of both qualitative and quantitative protein transporter analysis has been established using SCIEX TripleTOF® 6600 system.

References

1. Larissa B, etc. Membrane Protein Quantification by Peptide-Based Mass Spectrometry Approaches: Studies on the Organic Anion-Transporting Polypeptide Family, J Proteomics Bioinform. 2012;S4-003

2. Junichi K, etc. Quantitative Atlas of Membrane Transpoter Proteins:Development and Application of a High Sensitive Simulataneous LC/MS/MS Method Combined with Novel In-silico Peptide Selection Criteria, Pharmaceutical Research 2008; 25(6):1469- 1483.

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Trademarks/Licensing

For Research Use Only. Not for use in diagnostic procedures.

Document #: RUOMKT-02-1903

© 2015 AB Sciex. SCIEX is part of AB SCIEX. The trademarks mentioned herein are the property of AB Sciex Pte. Ltd. or their respective owners.

AB SCIEX™ is being used under license.

RUOMKT-02-1903