Top-down analysis of protein isoprenylation by electrospray ionization hybrid quadrupole time-of-flight tandem mass spectrometry; the mouse Tgamma protein.
Protein isoprenylation, an important post-translational modification with a lipid, involves the selective attachment of two types of isoprenoids, farnesyl (C15) and geranylgeranyl (C20). The isoprenoid is linked via a thioether bond to the C-terminal cysteine residue of a variety of cellular proteins, including the heterotrimeric G protein gamma-subunits. One member of the G protein family, transducin (Talpha/Tbetagamma), plays a central role in visual transduction, and the structure-function relationship has been extensively studied with purified proteins, predominantly with bovine transducin that was shown to be farnesylated at the C-terminal cysteine residue of the gamma-subunit (Tgamma). We report here the structure of the C-terminal modification of mouse Tgamma, which has not yet been elucidated owing to the low amount of protein that can be isolated from the mouse retina. Electrospray ionization mass spectrometry (ESI-MS) of the high-performance liquid chromatography (HPLC)-purified Tgamma was in good agreement with the calculated mass of the farnesylated and methylated form of mouse Tgamma (Pro1-Cys70). A 'top-down' analysis of intact Tgamma using an ESI hybrid quadrupole time-of-flight (TOF) tandem mass spectrometer provided isoprenyl-specific ions that were observed to produce ions separated by 204 Da from the conventional (unmodified) precursor ion or the C-terminal sequence ions. Such characteristic fragmentation on an isoprenoid observed in top-down analysis could be useful in general for determining the type of isoprenylation as well as probing the site of modification in the protein sequence.
Kassai
Hidetoshi
H
Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
Satomi
Yoshinori
Y
Fukada
Yoshitaka
Y
Takao
Toshifumi
T
D000818:Animals; D051379:Mice; D008810:Mice, Inbred C57BL; D017368:Protein Prenylation; D012374:Rod Cell Outer Segment / Q000737:chemistry*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000295:instrumentation / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000295:instrumentation / Q000379:methods*; D015293:Transducin / Q000032:analysis / Q000737:chemistry*
D015293:Transducin