title: Combining chymotrypsin/trypsin digestion to identify hydrophobic proteins from oil bodies.
abstract: Oil bodies, lipid-storage organelles, are stabilized by a number of specific proteins. These proteins are very hydrophobic, which complicates their identification by "classical" proteomic protocols using trypsin digestion. Due to the lack of trypsin cleavage sites, the achievable protein coverage is limited or even insufficient for reliable protein identification. To identify such proteins and to enhance their coverage, we introduced a modified method comprising standard three-step procedure (SDS-PAGE, in-gel digestion, and LC-MS/MS analysis). In this method, chymotrypsin, single or in combination with trypsin, was used, which enabled to obtain proteolytic peptides from the hydrophobic regions and to identify new oil bodies' proteins. Our method can be easily applied to identification of other hydrophobic proteins.
authors.affiliation: Department of Biochemistry and Microbiology, Institute of Chemical Technology Prague, Prague, Czech Republic.
authors.forename: Martina
authors.initials: M
authors.lastname: Vermachova
authors.forename: Zita
authors.initials: Z
authors.lastname: Purkrtova
authors.forename: Jiri
authors.initials: J
authors.lastname: Santrucek
authors.forename: Pascale
authors.initials: P
authors.lastname: Jolivet
authors.forename: Thierry
authors.initials: T
authors.lastname: Chardot
authors.forename: Milan
authors.initials: M
authors.lastname: Kodicek
mesh_terms: D017360:Arabidopsis / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000378:metabolism*; D002853:Chromatography, Liquid; D002918:Chymotrypsin / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D057927:Hydrophobic and Hydrophilic Interactions; D008055:Lipids; D013058:Mass Spectrometry; D015388:Organelles / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*; D014357:Trypsin / Q000378:metabolism*
chemical_list: D029681:Arabidopsis Proteins; D008055:Lipids; D002918:Chymotrypsin; D014357:Trypsin