Analysis of Cocaine Binding Site of Human DopamineTransporter Using Affinity Labeling and MassSpectrometry Público

Danilenko, Uliana Igorevna (2008)

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The small peptide PLFYM located in transmembrane domain two (TM2) of the human dopamine transporter (hDAT) is suggested to be the labeling site for the irreversible hDAT inhibitor [125I] MFZ 2-24. To locate the [125I] MFZ 2-24 labeling site, hDAT was photolabeled with [125I] MFZ 2-24 and digested with CNBr. The HPLC analysis of the digest showed that one small peptide was labeled. Parnas et al. (2003) demonstrated that [125I] MFZ 2-24 incorporates in transmembrane domains 1 and 2 of hDAT. The possible peptides from the CNBr digest include only two small hDAT peptides from the TM1-2 region, PLFYM and VIAGM, both in TM 2. The secondary enzymatic digests of the small labeled peptide from the CNBr digest of hDAT provided additional information supporting PLFYM being the labeled peptide (Wirtz, 2004). To further locate the [125I] MFZ 2-24 labeling site, hDAT was photolabeled with [125I] MFZ 2-24 and digested with thermolysin. The digest was separated on HPLC and the fraction corresponding to the labeled peptide collected. Edman degradation of the labeled peptide from the thermolysin digest of hDAT suggested that the second amino acid from the N-terminal was labeled, based on release of radioactivity. Additionally, labeled peptide from a separate thermolysin digest was re-digested with CNBr. The labeled peptide was analyzed by HPLC before and after the CNBr digest. The retention time of the [125I] MFZ 2-24 labeled peptide shifted, indicating that the peptide has a methionine in the sequence. The results from the CNBr and thermolysin digests in relation to the leucine transporter crystal structure (Yamashita et al., 2005) suggest that PLFYM is the labeled region, and F114 or Y115 are the labeled amino acid residues.

The hDAT mass spectrometry coverage and detection limit were examined using a ThermoFinnigan LTQ-FT high resolution mass spectrometer. The peptides from a thermolysin digest were separated on a nanoLC connected to a nanoelectrospray source. Mass spectrometry detection of hDAT peptides from the thermolysin digest resulted in 37 % coverage. The detected hDAT peptides were from the N-,C-terminal regions and the loop regions. A smaller number of peptides were also located in the transmembrane domain regions. This result supports mass spectrometry studies of membrane proteins that indicate the difficulties in detecting peptides from the transmembrane regions of membrane proteins (Wu et al., 2007; Yates et al., 2003). The detection limits for MFZ 2- 24 and for the synthetic hDAT peptide FYMELAL were found to be 5 fmol and 10 fmol respectively. Thus the 11.4 fmol of [125I] MFZ 2-24 labeled hDAT peptide left after in- gel digestion and extraction steps of 100 plates of HEK cells is near the detection limit of the instrument.

Table of Contents

Chapter One: Introduction

The Problem of Cocaine Abuse

hDAT Structure and Function

hDAT is a Membrane Protein

hDAT is a Member of the NSS Family

Overview of hDAT Function

Regulation of hDAT Function

Role of hDAT in Human Disease

hDAT Structure Overview

Dopamine Transporter Inhibitors

Mechanism of Cocaine Action

Overview of Different Classes of DAT Inhibitors

Investigation of Cocaine Binding Site

Affinity Labeling

Mutagenesis of DAT

Crystal Structure of Leucine Transporter

Mass Spectrometry

Mass Spectrometry of Membrane Proteins

Fourier Transform Ion Cyclotron Resonance Mass Spectrometry for Protein Analysis

Tandem Mass Spectrometry in Detection of Proteins and their Post-translational Modifications

Chapter Two: Methods

Cell Culture and Photoaffinity Labeling

Membrane Preparation

Photoaffinity Labeling

Gel Electrophoresis and Autoradiography

In-gel Chemical and Enzymatic Digests

Cyanogen Bromide Digest

Enzymatic Digests

High Performance Liquid Chromatography

HPLC Separation of hDAT Peptides

Radioactivity Profile Measurement

Sequencing of Labeled hDAT Peptides

The Manual Edman Degradation of Labeled hDAT Peptides

Chemical Labeling of Synthetic Peptides and Amino Acids

Chemical Labeling with MFZ 3-37

Examination of C90 as a Potential Labeling Site

3D Modeling of hDAT using the Spdbv Viewer

Labeling of hDAT and Mutant hDAT with [125I] MFZ 2-24 and [125I]MFZ 3-37

Incubation of hDAT and its Mutants with MTS Reagents

Mass Spectrometry Analysis of hDAT

Sample Preparation

Fabrication of In-house LC Nanocolumns

Determination of the Mass Spectrometer Sensitivity to Synthetic Peptides and MFZ 2-24

Overview of Mass Spectrometry Experiment

Data Analysis

hDAT Affinity Purification

Western Blot Analysis

hDAT Purification using FLAG M2 Affinity Chromatography

Purification of Labeled hDAT using MFZ 3-37 Antibodies

Chapter Three: Results

Analysis of [125I] MFZ 2-24 Labeled hDAT

WIN 35,428 Protection Experiment

CNBr Digest of [125I] MFZ 2-24 Labeled hDAT

Thermolysin Digest of [125I] MFZ 2-24 Labeled hDAT

Edman Degradation Analysis of the [125I] MFZ 2-24

Labeled hDAT Peptide

Effect of the Label on the Retention Time of Peptides and Amino Acids

Analysis of Cysteine 90 hDAT Mutant

Affinity Labeling of Wild Type and X5C hDAT

Role of C90 in hDAT Photoaffinity Labeling with [125I] MFZ 2-24

Role of C90 in hDAT Affinity Labeling with [125I] MFZ 3-37

Mass Spectrometry Analysis of hDAT

Mass Spectrometry Analysis of MFZ 2-24 and Synthetic Peptides

Sensitivity of FT-ICR Mass Spectrometer

Mass Spectrometry of 3xFLAG-6XHis-hDAT

Stability of MFZ 3-37 Reacted with hDAT Peptides

hDAT purification

Chapter Four: Discussion

Introduction to the Discussion

Investigation of hDAT Binding Site using [125I] MFZ 2-24 and [125I] MFZ 3-37

WIN 35,428 Protection Experiment

Analysis of [125I] MFZ 2-24 Labeled Peptide Resulting from CNBr Digest

Interpretation of Synthetic VIAGM and PLFYM Reacted with MFZ 3-37

Interpretation of the Thermolysin Digest Result

Interpretation of the Edman Degradation Result

Analysis of the Influence of MFZ 3-37 on the Retention Time of Synthetic Peptides and Amino Acids

TM1 and TM2 in Light of the LeuTAa Crystal Structure

Crystal Structure of a Member of the NSS Family, LeuTAa

Inhibitor Binding Site on the LeuTAa

Involvement of TM2 in Cocaine Binding

PLFYM as a Possible Region of hDAT Labeling

Involvement of TM1 in Cocaine Binding

Analysis of Cysteine 90 hDAT Mutant

C90 as a Possible Site of MFZ 2-24 and MFZ 3-37 Labeling

Interpretation of MFZ 2-24 and MFZ 3-37 Labeling of WT and X5C hDAT

Analysis of X-A90C hDAT Labeling in the Presence of Cysteine-Reactive MTS Reagents

Mass Spectrometry Approach to the Identification of hDAT Peptides and Labeled hDAT Peptides

Sample Preparation Strategies for Membrane Protein hDAT

Detection Limit for FT-ICR Mass Spectrometer Coupled with NanoLC Chromatography

Identification of hDAT Peptides from the Thermolysin Digest

Approach to Detect Labeled Peptides using NanoLC Nanospray FT-ICR Mass Spectrometer


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