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

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.