Analysis of Cocaine Binding Site of Human DopamineTransporter Using Affinity Labeling and MassSpectrometry Öffentlichkeit
Danilenko, Uliana Igorevna (2008)
Published
Abstract
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
References
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