This R21 application (10 pages max) is being submitted in response to PAR-03-017 """"""""Cutting-Edge Basic Research Awards (CEBRA)"""""""" which provides support for highly innovative or conceptually creative research to advance the understanding of drug abuse and addiction. ? ? Accurate assessments of the lipid embedded residues of membrane proteins are crucial for the prediction of membrane protein function and the generation of structural models. Currently, membrane protein topology prediction algorithms are accurate to +/- 5 to 10 amino acids at best. We propose to develop a shotgun proteomic approach for the global analysis of lipid embedded residues of integral membrane proteins within a complex biological sample. We will apply these methods to: 1) a directed approach to characterize the topology of the human dopamine transporter (hDAT), trafficking mutants of hDAT, and native DAT in rat brain tissue and 2) an undirected global approach to characterize the topology of membrane proteins enriched from cell and tissue homogenates. ? ? ?

Agency
National Institute of Health (NIH)
Institute
National Institute on Drug Abuse (NIDA)
Type
Exploratory/Developmental Grants (R21)
Project #
1R21DA021744-01
Application #
7135141
Study Section
Special Emphasis Panel (ZDA1-MXS-M (20))
Program Officer
Colvis, Christine
Project Start
2006-05-01
Project End
2008-04-30
Budget Start
2006-05-01
Budget End
2007-04-30
Support Year
1
Fiscal Year
2006
Total Cost
$152,380
Indirect Cost
Name
University of Colorado Denver
Department
Pharmacology
Type
Schools of Medicine
DUNS #
041096314
City
Aurora
State
CO
Country
United States
Zip Code
80045
Farias, Santiago E; Kline, Kelli G; Klepacki, Jacek et al. (2010) Quantitative improvements in peptide recovery at elevated chromatographic temperatures from microcapillary liquid chromatography-mass spectrometry analyses of brain using selected reaction monitoring. Anal Chem 82:3435-40
Blackler, Adele R; Speers, Anna E; Ladinsky, Mark S et al. (2008) A shotgun proteomic method for the identification of membrane-embedded proteins and peptides. J Proteome Res 7:3028-34
Blackler, Adele R; Speers, Anna E; Wu, Christine C (2008) Chromatographic benefits of elevated temperature for the proteomic analysis of membrane proteins. Proteomics 8:3956-64
Finney, Gregory L; Blackler, Adele R; Hoopmann, Michael R et al. (2008) Label-free comparative analysis of proteomics mixtures using chromatographic alignment of high-resolution muLC-MS data. Anal Chem 80:961-71
Speers, Anna E; Blackler, Adele R; Wu, Christine C (2007) Shotgun analysis of integral membrane proteins facilitated by elevated temperature. Anal Chem 79:4613-20
Speers, Anna E; Wu, Christine C (2007) Proteomics of integral membrane proteins--theory and application. Chem Rev 107:3687-714