Protein prenylation in an insect cell-free protein synthesis system and identification of products by mass spectrometry.
To evaluate the ability of an insect cell-free protein synthesis system to carry out proper protein prenylation, several CAIX (X indicates any C-terminal amino acid) sequences were introduced into the C-terminus of truncated human gelsolin (tGelsolin). Tryptic digests of these mutant proteins were analyzed by MALDI-TOF MS and MALDI-quadrupole-IT-TOF MS. The results indicated that the insect cell-free protein synthesis system possesses both farnesyltransferase (FTase) and geranylgeranyltransferase (GGTase) I, as is the case of the rabbit reticulocyte lysate system. The C-terminal amino acid sequence requirements for protein prenylation in this system showed high similarity to those observed in rat prenyltransferases. In the case of rhoC, which is a natural geranylgeranylated protein, it was found that it could serve as a substrate for both prenyltransferases in the presence of either farnesyl or geranylgeranyl pyrophosphate, whereas geranylgeranylation was only observed when both prenyl pyrophosphates were added to the in vitro translation reaction mixture. Thus, a combination of the cell-free protein synthesis system with MS is an effective strategy to analyze protein prenylation.
Suzuki
Takashi
T
Life Science Laboratory, Analytical and Measuring Instruments Division, Shimadzu Corporation, Kyoto, Japan. t-suzuki@shimadzu.co.jp
Ito
Masaaki
M
Ezure
Toru
T
Shikata
Masamitsu
M
Ando
Eiji
E
Utsumi
Toshihiko
T
Tsunasawa
Susumu
S
Nishimura
Osamu
O
D019883:Alkyl and Aryl Transferases / Q000378:metabolism*; D000818:Animals; D002457:Cell Extracts; D003001:Cloning, Molecular; D051231:Farnesyltranstransferase / Q000378:metabolism*; D018260:Gelsolin / Q000378:metabolism*; D006801:Humans; D019476:Insect Proteins / Q000378:metabolism; D007313:Insecta / Q000378:metabolism; D017368:Protein Prenylation; D011817:Rabbits; D051381:Rats; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D020741:rho GTP-Binding Proteins / Q000378:metabolism
D002457:Cell Extracts; D018260:Gelsolin; D019476:Insect Proteins; D019883:Alkyl and Aryl Transferases; C083291:geranylgeranyltransferase type-I; D051231:Farnesyltranstransferase; D020741:rho GTP-Binding Proteins