![]() ![]() Genomic studies have pinpointed several single mutations in GABA AR as the causes-and potential drug targets-of epilepsy, a devastating disorder that afflicts 2.2 million Americans and 65 million people worldwide at tremendous health and economic costs ( 4 – 12). Formed by five homologous TM subunits (main form in brain (α1) 2(β2/β3) 2(γ2L) 1) ( 2, 3), Cys-loop LGIC gamma-aminobutyric acid type A receptor (GABA AR) is the major inhibitory (C l ̄) ion channel that balances excitation in CNS. Ion channels already represent 10% of current drug targets before the structure-function mechanisms of CNS LGICs become clear ( 1). TM proteins such as ligand-gated ion channels (LGICs) and G protein-coupled receptors (GPCRs) are cell gate-keepers that convert external signals into cellular activities throughout the central nervous system (CNS), and predominate as desired drug targets. About 25% of the 29,375 unique protein sequences in human proteome contain at least one TM alpha-helix, and 13% contain at least two ( 1). Transmembrane (TM) 1 proteins are abundant and play critical roles in nearly all biological processes. These results enabled streamlined proteomics of human synaptic membrane proteins, and more importantly, allowed directly coupling proteomics with crystallography to characterize both static and dynamic structures of membrane proteins in crystallization pipelines. The purification conditions were comparable to those applied in successful crystallizations of most membrane proteins. Sonication or dropwise addition of detergent robustly solubilized over 90% of membrane pellets. Unlike conventional belief that detergents must be removed before HPLC MS, the high-purity low-dose nonionic detergent DDM did not interfere with peptides, and obviated removal or desalting. Using 285-kDa hetero-pentameric human GABA type A receptor overexpressed in HEK293 as a model, we describe a n-dodecyl-β- d-maltopyranoside/cholesteryl hemisuccinate (DDM/CHS)-based affinity purification method, that produced active receptors, supported protease activity, and allowed high performance with both in-gel and direct gel-free proteomic analyses-without detergent removal. Contrary to attempts to subdue, we resolved this challenge by providing proteins nature-and-activity-promoting conditions throughout preparation. Traditional protein extraction methods apply ionic or poly-disperse detergents, harsh denaturation, and repeated protein/peptide precipitation/resolubilization afterward, but suffer low yield, low reproducibility, and low sequence coverage. The challenge in high-quality membrane proteomics is all about sample preparation prior to HPLC, and the cell-to-protein step poses a long-standing bottleneck.
0 Comments
Leave a Reply. |
Details
AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |