Suzuki Takashi, Ito Masaaki, Ezure Toru, Shikata Masamitsu, Ando Eiji, Utsumi Toshihiko, Tsunasawa Susumu, Nishimura Osamu
Life Science Laboratory, Analytical and Measuring Instruments Division, Shimadzu Corporation, Kyoto, Japan.
Proteomics. 2007 Jun;7(12):1942-50. doi: 10.1002/pmic.200700237.
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.
为了评估昆虫无细胞蛋白质合成系统进行正确蛋白质异戊二烯化的能力，将几个CAIX（X表示任何C末端氨基酸）序列引入截短的人凝溶胶蛋白（tGelsolin）的C末端。通过MALDI-TOF MS和MALDI-四极杆-离子阱-TOF MS分析这些突变蛋白的胰蛋白酶消化产物。结果表明，昆虫无细胞蛋白质合成系统同时具有法尼基转移酶（FTase）和香叶基香叶基转移酶（GGTase）I，兔网织红细胞裂解物系统也是如此。该系统中蛋白质异戊二烯化的C末端氨基酸序列要求与在大鼠异戊二烯转移酶中观察到的要求高度相似。对于天然香叶基香叶基化蛋白rhoC，发现在存在法尼基焦磷酸或香叶基香叶基焦磷酸的情况下，它都可以作为两种异戊二烯转移酶的底物，而仅当将两种异戊二烯焦磷酸都添加到体外翻译反应混合物中时才观察到香叶基香叶基化。因此，将无细胞蛋白质合成系统与质谱联用是分析蛋白质异戊二烯化的有效策略。
