A Proteomics Approach to the Identification of Mammalian Mitochondrial Small Subunit Ribosomal Proteins
peer-reviewed · Journal of Biological Chemistry · 2000
| Date | 2000-10-01 |
| Type | peer-reviewed |
| Venue | Journal of Biological Chemistry |
| Publisher | American Society for Biochemistry and Molecular Biology |
| Contribution | downstream-application |
| DOI | 10.1074/jbc.M003596200 |
Abstract
Mammalian mitochondrial small subunit ribosomal proteins were separated by two-dimensional polyacrylamide gel electrophoresis. The proteins in six individual spots were subjected to in-gel tryptic digestion. Peptides were separated by capillary liquid chromatography, and the sequences of selected peptides were obtained by electrospray tandem mass spectrometry. The peptide sequences obtained were used to screen human expressed sequence tag data bases, and complete consensus cDNAs were assembled. Mammalian mitochondrial small subunit ribosomal proteins from six different classes of ribosomal proteins were identified. Only two of these proteins have significant sequence similarities to ribosomal proteins from prokaryotes. These proteins correspond to Escherichia coli S10 and S14. Homologs of two human mitochondrial proteins not found in prokaryotes were observed in the genomes of Drosophila melanogaster and Caenorhabditis elegans. A homolog of one of these proteins was observed in D. melanogaster but not in C. elegans, while a homolog of the other was present in C. elegans but not in D. melanogaster. A homolog of one of the ribosomal proteins not found in prokaryotes was tentatively identified in the yeast genome. This latter protein is the first reported example of a ribosomal protein that is shared by mitochondrial ribosomes from lower and higher eukaryotes that does not have a homolog in prokaryotes.
Methods and tools
- Manual MS/MS de novo interpretation: Protocol for manual de novo peptide sequencing from MS/MS spectra.
- Mitochondrial small subunit ribosome proteomics: 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.
- PepSeq: Interactive de novo peptide sequencing application in BioLynx, the biopolymer analysis component of the MassLynx suite. The manual is explicit that it is user-driven rather than automatic: PepSeq “has been designed as an interactive program that allows the user to make decisions at each step in deducing the sequence of a peptide”. It scores and annotates spectra on the a, b, y and z ions, and takes input either from a MassLynx data file or as a plain mass and intensity list. Three modes: typing a candidate sequence to see its theoretical fragments annotated against the spectrum; FindTag, which builds a set of sub-sequences whose series ions best match the spectrum and extends them step or leap-wise until the molecular weight matches the user-supplied precursor; and MassSeq, a separately purchased option that performs the sequencing automatically from the precursor mass, modifications and a mass-accuracy estimate. There is no methods paper: the method is documented only in the vendor manual, which is catalogued here as the accompanying resource. Provenance runs Micromass UK Ltd to Waters Corporation, and the Version 4.0 guide catches the handover in progress, carrying a Waters part number and a Micromass part number side by side over a 1993-2002 Micromass UK Ltd copyright.