A fully automated phosphopeptide purification system for large-scale phosphoproteome analysis.

For large-scale phosphoproteome analysis based on mass spectrometry, a fully automated phosphopeptide purification system is essential to obtain reproducible results. An automated system involving pre-cleaning of a sample with a polymer-based reversed-phase column, phosphopeptide purification with a titania column and analysis of the phosphopeptide fraction with a reversed-phase column was developed, and then the analytical conditions for a complex peptide mixture were optimized. A lower flow rate for application of samples to the titania column was essential to obtain high recoveries of phosphopeptides from complex protein digests. Washing with 1 M NaCl and 2-propanol, and two cycles of washing with four solvents for the titania column were necessary to minimize non-phosphorylated peptides in the phosphopeptide fraction. Using this system under the optimized conditions, a peptide fraction including >90% phosphopeptides could be obtained highly reproducibly from a tryptic digest of a complex protein mixture, i.e. a Xenopus egg cytosol fraction, without any pre-treatment.

Evolution from possible primitive tRNA-viroids to early poly-tRNA-derived mRNAs: a new approach from the poly-tRNA theory.

Poly-tRNA theory have revealed that the tRNA gene-clusters in the Bacillus subtilis trrnD- and rrnB-operons are relics of early peptide-synthesizing RNA apparatus. The trrnD-type and rrnB-type poly-tRNA models were re-analyzed by using recent databases. The results elucidated that the 16 amino acid (aa)-trrnD- and the 21 aa-rrnB-peptides (whose aa sequences are in the order of aa specificities of tRNAs in the respective tRNA cluster) are really relics of earliest peptides encoded by most primitive mRNAs, trrnD-mRNA and rrnB-mRNA, which are homologous to tRNA(Gly) and tRNA(His), respectively. Genes encoding various protein superfamilies (including pgtB protein, glycyl-tRNA synthetase alpha, C-type lectin, F1-ATP-synthase gamma, etc) were concluded to have derived from tRNA(Gly)-tRNA(Cys)-tRNA(Leu) region (including trrnD-mRNA region) in the trrnD-poly-tRNA. Genes for another group of protein superfamilies (including adenylate kinase, glyceraldehyde-3-phosphate dehydrogenase, helix-turn-helix DNA-binding domains, etc.) were found to have derived rrnB-mRNA, which is most plausibly homologous to a region containing the tRNA(His) of the rrnB-poly-tRNA. Thus Proto-tRNA((Gly)) reconstructed from tRNA(Gly) and other tRNAs strongly suggested that proto-tRNA was most plausibly a viroid-like possibly self-cleavable replicable ribozyme possessing a possible hammerhead-like structure.

Origin of most primitive mRNAs and genetic codes via interactions between primitive tRNA ribozymes.

The origin and early evolution of genetic codon system and early mRNAs were analyzed from a viewpoint of primordial gene theory and the poly-tRNA theory. A hypothetical 25-amino acid (aa)-primordial peptide was deduced from internal aa-sequence homology of adenylate kinases. Theoretical models were made which can reasonably explain how primitive tRNA(s) could have had converted to be earliest mRNAs via interactions between presumptive anticodons and (poly-)tRNA ribozyme. Transfer-RNA gene clusters in the trrnD- and rrnB-operons of Bacillus subtilis seemed to be relics of early peptide-synthesizing RNA machine. Detailed analyses revealed that the poly-tRNA regions in these operons are true relics of RNA-machine for making a 16-aa trrnD-peptide and a 21-aa rrnB-peptide, whose aa sequences are in the order of aa specificities of tRNAs in the tRNA gene clusters of the trrnD-operon and rrnB-operon, respectively. The primordial gene-encoded peptide deduced from adenylate kinases were found to be a genuine homologue of the rrnB-peptide. Various protein superfamilies were found to have evolved from either of these two types of primitive peptides. Earliest mRNAs were concluded to have evolved from tRNAGly (trrnD-mRNA) or tRNAHis (rrnB-mRNA), where trrnD- and rrnB-mRNAs are hypothetical primitive mRNAs complementary to the tandem arrangement of 16 or 21 anticodons of tRNAs in the trrnD-operon and rrnB-operon, respectively. The poly-tRNA model is considered to be an excellent theory, because it can reasonably explain origins of both genetic codes and earliest mRNAs, and because the hypothesis can be statistically evaluated by base-identity levels in proper alignments. The genetic codon system is a typical mature semeiotic system within a cell, and the genesis of the genetic codon system was discussed from an aspect of de Saussure's semeiology. Arbitrary correspondence between (anti)codon and aa would be most plausibly a result of semeiotic culture system of intracellular tRNA-riboorganismic society.