N-Terminal protein modifications in an insect cell-free protein synthesis system and their identification by mass spectrometry Export

@article{citeulike:3889188, abstract = {To evaluate the ability of an insect cell-free protein synthesis system to generate proper N-terminal cotranslational protein modifications such as removal of the initiating Met, N-acetylation, and N-myristoylation, several mutants were constructed using truncated human gelsolin (tGelsolin) as a model protein. Tryptic digests of these mutants were analyzed by MALDI-TOF MS and MALDI-quadrupole-IT-TOF MS. The wild-type tGelsolin, which is an N-myristoylated protein, was found to be N-myristoylated when myristoyl-CoA was added to the in vitro translation reaction mixture. N-myristoylation did not occur on the Gly-2 to Ala mutant, in which the N-myristoylation motif was disrupted, whereas this mutant was found to be N-acetylated after removal of the initiating Met. Analyses of Gly-2 to His and Leu-3 to Asp mutants revealed that the amino acids at positions 2 and 3 strongly affect the susceptibility of the nascent peptide chain to removal of the initiating Met and to N-acetylation, respectively. These results suggest that N-terminal modifications occurring in the insect cell-free protein synthesis system are quite similar to those observed in the mammalian protein synthesis system. Thus, a combination of the cell-free protein synthesis system with MS is an effective strategy to analyze protein modifications.}, address = {Life Science Laboratory, Analytical and Measuring Instruments Division, Shimadzu Corporation, Kyoto, Japan; Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University, Yamaguchi, Japan; The Research Center for Structural and Functional Proteomics, Institute for Protein Research, Osaka University, Osaka, Japan}, author = {Suzuki, Takashi and Ito, Masaaki and Ezure, Toru and Shikata, Masamitsu and Ando, Eiji and Utsumi, Toshihiko and Tsunasawa, Susumu and Nishimura, Osamu}, citeulike-article-id = {3889188}, citeulike-linkout-0 = {http://dx.doi.org/10.1002/pmic.200600126}, citeulike-linkout-1 = {http://www3.interscience.wiley.com/cgi-bin/abstract/112696176/ABSTRACT}, doi = {10.1002/pmic.200600126}, journal = {PROTEOMICS}, keywords = {acetylation, methods}, number = {16}, pages = {4486--4495}, posted-at = {2009-01-15 16:41:51}, priority = {2}, title = {N-Terminal protein modifications in an insect cell-free protein synthesis system and their identification by mass spectrometry}, url = {http://dx.doi.org/10.1002/pmic.200600126}, volume = {6}, year = {2006} }

TY - JOUR ID - citeulike:3889188 L3 - citeulike-article-id:3889188 N2 - To evaluate the ability of an insect cell-free protein synthesis system to generate proper N-terminal cotranslational protein modifications such as removal of the initiating Met, N-acetylation, and N-myristoylation, several mutants were constructed using truncated human gelsolin (tGelsolin) as a model protein. Tryptic digests of these mutants were analyzed by MALDI-TOF MS and MALDI-quadrupole-IT-TOF MS. The wild-type tGelsolin, which is an N-myristoylated protein, was found to be N-myristoylated when myristoyl-CoA was added to the in vitro translation reaction mixture. N-myristoylation did not occur on the Gly-2 to Ala mutant, in which the N-myristoylation motif was disrupted, whereas this mutant was found to be N-acetylated after removal of the initiating Met. Analyses of Gly-2 to His and Leu-3 to Asp mutants revealed that the amino acids at positions 2 and 3 strongly affect the susceptibility of the nascent peptide chain to removal of the initiating Met and to N-acetylation, respectively. These results suggest that N-terminal modifications occurring in the insect cell-free protein synthesis system are quite similar to those observed in the mammalian protein synthesis system. Thus, a combination of the cell-free protein synthesis system with MS is an effective strategy to analyze protein modifications. IS - 16 JF - PROTEOMICS AD - Life Science Laboratory, Analytical and Measuring Instruments Division, Shimadzu Corporation, Kyoto, Japan; Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University, Yamaguchi, Japan; The Research Center for Structural and Functional Proteomics, Institute for Protein Research, Osaka University, Osaka, Japan EP - 4495 TI - N-Terminal protein modifications in an insect cell-free protein synthesis system and their identification by mass spectrometry VL - 6 SP - 4486 KW - acetylation KW - methods AU - Suzuki, Takashi AU - Ito, Masaaki AU - Ezure, Toru AU - Shikata, Masamitsu AU - Ando, Eiji AU - Utsumi, Toshihiko AU - Tsunasawa, Susumu AU - Nishimura, Osamu PY - 2006/// UR - http://dx.doi.org/10.1002/pmic.200600126 ER -