Rubisco large subunit N-terminal determination
downstream-application
De novo sequencing used to establish what the N terminus of a recombinant protein actually is, which the biological argument then rests on. Arabidopsis Rubisco large subunit variants expressed in E. coli are processed unpredictably by the host, so the construct sequence does not tell you which residues survive. Purified proteins were run on SDS gels, the RbcL band excised, and the N termini determined by de novo mass spectrometric sequencing in PEAKS Studio X+, alongside a semi-specific tryptic database search and peak-area quantification; the work was done as a service by the Bioprocessing Technology Institute, A*STAR. The result is Table 1 in full: the observed N termini for wild type and each truncation variant with their peak-area ratios, showing for instance that wild type is a mixture of MSPQTETKAS, SPQTETKAS and PQTETKAS at 36, 15 and 49 percent. Those assignments are what let the paper claim that large subunits lacking residues 1 to 4 are functional carboxylases that Rubisco activase cannot activate, and they drive a further inference about cooperativity, since some deltaN2 N termini proved identical to deltaN3 ones. A tryptic database search alone would not have found them, because host processing leaves ragged non-tryptic N termini.
| Kind | downstream-application |
| Deep learning | no |
| Acquisition | DDA |
| Application area | general-proteomics |
Papers
- Rubisco activase requires residues in the large subunit N terminus to remodel inhibited plant Rubisco (2020, Journal of Biological Chemistry, peer-reviewed)