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

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.

Expression of human Cu, Zn-superoxide dismutase in an insect cell-free system and its structural analysis by MALDI-TOF MS

“…Whereas the expression and characterization of plant-specific zinc-binding transcriptional factors have been well investigated, only the expression of the DNA binding domains, but not of the entire mature proteins, has been described. A cell-free protein synthesis system (Transdirect insect cell) derived from Sf21 insect cells (Ezure et al, 2006;Suzuki et al, 2006a) has been developed as a tool for post-genomic studies to improve the efficiency of producing targeted proteins, especially in cases where it is difficult to obtain sufficient amounts for analyses, including measurement of enzymatic activity, western blotting and investigation of post-translational modifications such as N-terminal protein modifications (Suzuki et al, 2006b), protein prenylation and formation of disulfide bonds by MS. Other post-translational modifications such as formation of protein complexes (Masuda et al, 2007) and ubiquitination (to be published elsewhere) also occurred in the insect cell-free system. In addition, core glycosylation and cleavages of signal peptides were observed after the addition of microsomal membranes to the reaction mixture (unpublished data).…”

The Protein Lipidation and Its Analysis

“…Similarly, the heterogenicity of the lipid groups present in the GAP-43 protein, which has two acylated cysteines near the C-terminus, was studied by MALDI-TOF and confirmed by Quadropole-TOF MS/MS indicating that not only palmitoyl but also stearic acid can be attached on these cysteines [51]. Recently, MALDI-MS was also used for the detection of purified myristoylated and isoprenylated proteins generated in an insect-cell free protein synthesis system [88][89][90]. However, one of the limitations of this approach is the high hydrophobicity of the lipidated samples, which usually results in low efficiency of peptide recovery during sample preparation as well as peptide detection.…”

“…The resulting peptide fragments are then analyzed by mass spectrometry (either ESI-MS or MALDI-MS) and identified by peptide-mass fingerprint. A classic example of this approach is the detection of the palmitoylated Shh protein using an HPLC method coupled to a triple quadruple mass spectrometer, followed by confirmation by sequencing of the lipidated N-terminal peptide by MALDI [88]. Similarly, the heterogenicity of the lipid groups present in the GAP-43 protein, which has two acylated cysteines near the C-terminus, was studied by MALDI-TOF and confirmed by Quadropole-TOF MS/MS indicating that not only palmitoyl but also stearic acid can be attached on these cysteines [51].…”

Preparation ofN-Acylated Proteins Modified with Fatty Acids Having a Specific Chain Length Using an Insect Cell-Free Protein Synthesis System

“…9) Using this system, N-myristoylated protein could be generated only when myristoyl-CoA was added to the reaction mixture. 8) From this observation, it was anticipated that acylated proteins having fatty acids other than myristic acid might be generated when the respective acyl-CoA species were added to the reaction mixture. To test this possibility, that the Sf21 insect cellfree protein synthesis system should be able to generate an arbitrary N-acylation corresponding to the added fatty acyl-CoA substrate species, various saturated unbranched fatty acyl-CoA esters with carbon chains of various lengths, such as octanoyl-(C 8 ), decanoyl-(C 10 ), lauroyl-(C 12 ), myristoyl-(C 14 ), palmitoyl-(C 16 ), and stearoyl-(C 18 ) CoA, were added individually to the translation reaction mixture, and in vitro translation reactions were performed.…”

“…The tryptic digests were analyzed by matrix-assisted laser desorption/ionizationtime of flight mass spectrometry (MALDI-TOF MS) in reflectron positive ion mode with an AXIMA-CFRÔ-plus MALDI TOF MS instrument and an AXIMA-QITÔ MALDI quadrupole ion trap (QIT) TOF hybrid mass spectrometer (Shimadzu/Kratos, Manchester, UK). 8) We have developed a cell-free protein synthesis y To whom correspondence should be addressed. Fax: +81-75-823-1364; E-mail: t-suzuki@shimadzu.co.jp Abbreviations: TNF, tumor necrosis factor; tGelsolin, truncated gelsolin; MALDI-TOF MS, matrix-assisted laser desorption/ionization-time of flight mass spectrometry; QIT, quadrupole ion trap; Sf21, Spodoptera frugiperda 21; m=z, mass-to-charge ratio; NMT, N-myristoyltransferase system derived from Spodoptera frugiperda 21 (Sf21) insect cells.…”