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Dissertation Information for Brian Reed

NAME:
- Brian Reed

DEGREE:
- Ph.D.

DISCIPLINE:
- Chemistry

SCHOOL:
- Emory University (USA) (2001)

ADVISORS:
- Joseph B. Justice Jr.

COMMITTEE MEMBERS:
- Vincent P. Conticello
- Dale E. Edmondson

MPACT Status: Fully Complete

Title: Photoaffinity labeling and peptide mapping of the human dopamine transporter

Abstract: "The compound para-azidophenethylamine (PAPEA) was designed to be both a substrate and photoaffinity label of the dopamine transporter (DAT). Upon synthesis of this compound, it was shown, using rotating disk electrode voltammetry, to be transported via DAT across the plasma membrane of recombinant HEK-293 cells. PAPEA was also capable of competing with [ 3 H]WIN 35,428 binding to the dopamine transporter in membrane preparations from the recombinant HEK-293 cell line. The photoreactive phenylazide moiety of PAPEA resulted in covalent labeling upon irradiation, as determined by irreversible inhibition of subsequent [ 3 H]WIN 35,428 binding to the dopamine transporter. The remainder of the present work represents the development of methodology for the localization of the particular amino acid residue(s) labeled by PAPEA, as well as those labeled by other irreversible labels of DAT.

The methodology for high-performance liquid chromatography (HPLC) separation coupled with mass spectral identification and sequencing of peptides resulting from digestion of integral membrane proteins was developed using bacteriorhodopsin as a model protein. Methionine-specific cyanogen bromide digestion of bacteriorhodopsin (bR) results in peptides of varying size and hydrophobicity. HPLC allowed for the partial resolution of the nine bR peptides. Nanoelectrospray ionization, with magnetic sector mass spectrometry, allowed for the identification of all peptides, and tandem mass spectrometry was used for the complete determination of sequence of five of the peptides, and partial determination of sequence of two of the larger peptides.

The application of mass spectral peptide mapping requires prior purification of the protein. To that end, the cDNA encoding DAT was incorporated into a vector encoding a 6x-polyhistidine tag. This altered gene encoding a histidine-tagged DAT (HisDAT) was expressed in a mammalian cell line (BEK-293), for purification efforts. The membranes were isolated, and the membrane proteins solubilized by treatment with detergents, typically Triton X-100. The solubilized DAT was then subsequently partially purified using immobilized metal affinity chromatography. Using [ 125 I]RTI-82, a DAT selective photoaffinity label, as a marker for DAT, gel electrophoresis followed by in-gel digestion with the proteolytic enzyme chymotrypsin and subsequent electrophoresis using high percentage acrylamide gels (15.5%), a peptide fragment comprising most of the 5 th transmembrane domain of DAT was detected using matrix-assisted laser desorption/ionization mass spectrometry."

MPACT Scores for Brian Reed

A = 0
C = 0
A+C = 0
T = 0
G = 0
W = 0
TD = 0
TA = 0
calculated 2008-04-07 08:16:29

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