General proteomics
3 workflows · 2000–2020
General proteomics: Ordinary discovery proteomics, where de novo sequencing fills in what the database search could not explain. 3 catalogued workflows and 3 papers.
Ordinary discovery proteomics, where de novo sequencing fills in what the database search could not explain.
| Workflows | 3 |
| Papers | 3 |
| Authors | 11 |
| Active | 2000-10-01 to 2020-11-01 |
Workflows (3)
- Mitochondrial small subunit ribosome proteomics (2000): Bovine mitochondrial small subunit ribosomal proteins resolved by two-dimensional PAGE, in-gel tryptic digestion, capillary LC and electrospray MS/MS, with the resulting peptide sequences used as virtual probes to screen the human EST database by tBLASTN and assemble consensus cDNAs in silico. Spectra without an exact match in either the protein or EST databases were sequenced de novo, manually or with PepSeq. Seven proteins are reported in Table I, and the two de novo-derived peptides are the sole identifying evidence for two of them, MRP-S26 and MRP-S14; MRP-S14 is also one of only two proteins in the study with significant prokaryotic homology, to Escherichia coli S14. Five of the seven belong to a new class of ribosomal proteins with no prokaryotic counterpart.
- Large-scale de novo application to complex proteomics (2016): Application of de novo sequencing to large-scale complex proteomics data sets.
- Rubisco large subunit N-terminal determination (2020): 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.
Papers (3)
- A Proteomics Approach to the Identification of Mammalian Mitochondrial Small Subunit Ribosomal Proteins (2000, Journal of Biological Chemistry, peer-reviewed)
- Application of de Novo Sequencing to Large-Scale Complex Proteomics Data Sets (2016, Journal of Proteome Research, peer-reviewed)
- Rubisco activase requires residues in the large subunit N terminus to remodel inhibited plant Rubisco (2020, Journal of Biological Chemistry, peer-reviewed)
Sequencing tools these papers used
Where the work happened
Singapore, USA