Top‐down analysis of protein isoprenylation by electrospray ionization hybrid quadrupole time‐of‐flight tandem mass spectrometry; the mouse Tγ protein

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 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.

Toward an “omic” physiopathology of reactive chemicals: Thirty years of mass spectrometric study of the protein adducts with endogenous and xenobiotic compounds

“…The nature of the cysteine-modifying group was inferred both by mass shift of the Y-ion series by 204u (the mass of the appended hydrocarbon chain) and by observation of loss of the same unit from the doubly protonated peptide (Heilmeyer et al, 1992). The same mass spectrometric behavior was observed in the characterization a farnesylated G-protein from the rat's visual system, both for the collision activated decomposition of a high charge state of the intact protein and for a C-terminal pentapeptide derived from V8-peptidase hydrolysis (Kassai et al, 2005), as well as for peptides carrying an internal, rather than C-terminal prenylated cysteine residue (Suzuki et al, 2007).…”

Mass spectrometric characterization of lipid‐modified peptides for the analysis of acylated proteins

“…Furthermore, as each fragment differs by the mass of a CH 2 unit, other ions seen in the product ion spectra that are multiples of 14 Da lower than C 10 H 15 C or 14 Da higher than C 12 H 19 C may be associated farnesyl fragment ions. Only one of these three fragments was previously identified by Kassai et al 30 as a fragment of the farnesyl moiety (149.1 Th), while the other fragment that they attributed to a farnesyl fragment (137.1 Th) seems erroneous. A high mass accuracy product ion analysis with a quadrupole TOF was then completed to resolve interfering ions with the same nominal mass as the farnesyl marker ion and associated fragment ions.…”

“…They also noted that in the low-mass region of the mass spectrum there was an ion of m/z 205.2 characteristic of the farnesyl moiety and attributed peaks at m/z 137.1 and m/z 149.1 to isoprenyl fragment ions. 30 Fragmentation of the singly- (Fig. 4(a), (b)), doubly- (Fig.…”

“…As well, they identified the charged fragment ion of m/z 205 corresponding to a farnesyl moiety with a proton. 30 Lastly, in the analysis of palmitoylation, researchers have used the mass shift in the MS spectra, identifying the modified from the unmodified protein or peptide, as well as the mass shift in the MS/MS spectra to identify the cysteine palmitoylation 31 and the rare N-terminal glycine palmitoylation. 32 As well, a marker ion was found for the rare lysine palmitoylation, although it is specific only for lysine palmitoylation.…”

Post-translational modifications and their applications in eye research

“…A similar top-down proteomic study of the isoprenylation of transducin examined the rod outer segment membrane of mice. In this study, similar modification sites were observed on murine Tγ when compared with those in bovine Tγ (187). Using the proteomic PTM approach, a high heterogeneic pattern of glycosylation on the 5-hydroxytryptamine receptor 4 (5-HT 4 R) in 5-HT 4 R-containing rod cells was discovered in transgenic mice (188).…”