Premium
Top‐down analysis of protein isoprenylation by electrospray ionization hybrid quadrupole time‐of‐flight tandem mass spectrometry; the mouse T γ protein
Author(s) -
Kassai Hidetoshi,
Satomi Yoshinori,
Fukada Yoshitaka,
Takao Toshifumi
Publication year - 2004
Publication title -
rapid communications in mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.528
H-Index - 136
eISSN - 1097-0231
pISSN - 0951-4198
DOI - 10.1002/rcm.1782
Subject(s) - chemistry , transducin , electrospray ionization , tandem mass spectrometry , mass spectrometry , cysteine , prenylation , protein subunit , biochemistry , stereochemistry , chromatography , g protein , signal transduction , enzyme , gene
Abstract 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 γ ‐subunits. One member of the G protein family, transducin (T α /T βγ ), 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 γ ‐subunit (T γ ). We report here the structure of the C‐terminal modification of mouse T γ , 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 T γ was in good agreement with the calculated mass of the farnesylated and methylated form of mouse T γ (Pro1‐Cys70). A ‘top‐down’ analysis of intact T γ 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. Copyright © 2004 John Wiley & Sons, Ltd.