Differential detergent fractionation for non-electrophoretic eukaryote cell proteomics.
Differential detergent fractionation (DDF), which relies on detergents to sequentially extract proteins from eukaryotic cells, has been used to increase proteome coverage of 2D-PAGE. Here, we used DDF extraction in conjunction with the nonelectrophoretic proteomics method of liquid chromatography and electrospray ionization tandem mass spectrometry. We demonstrate that DDF can be used with 2D-LC ESI MS2 for comprehensive cellular proteomics, including a large proportion of membrane proteins. Compared to some published methods designed to isolate membrane proteins specifically, DDF extraction yields comprehensive proteomes which include twice as many membrane proteins. Two-thirds of these membrane proteins have more than one trans-membrane domain. Since DDF separates proteins based upon their physicochemistry and subcellular localization, this method also provides data useful for functional genome annotation. As more genome sequences are completed, methods which can aid in functional annotation will become increasingly important.
McCarthy
Fiona M
FM
College of Veterinary Medicine, PO Box 6100, Mississippi State University, Mississippi 39762, USA.
Burgess
Shane C
SC
van den Berg
Bart H J
BH
Koter
Marek D
MD
Pharr
G Todd
GT
D000818:Animals; D002645:Chickens; D002853:Chromatography, Liquid; D003902:Detergents / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D005057:Eukaryotic Cells / Q000737:chemistry; D011506:Proteins / Q000302:isolation & purification*; D040901:Proteomics; D021241:Spectrometry, Mass, Electrospray Ionization
D003902:Detergents; D011506:Proteins