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

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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. © 2006 Wiley-VCH Verlag GmbH & Co. KGaA.

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Suzuki, T., Ito, M., Ezure, T., Shikata, M., Ando, E., Utsumi, T., … Nishimura, O. (2006). N-terminal protein modifications in an insect cell-free protein synthesis system and their identification by mass spectrometry. Proteomics, 6(16), 4486–4495. https://doi.org/10.1002/pmic.200600126

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