Characterization of the 70S Ribosome from Rhodopseudomonas palustris using an integrated "top-down" and "bottom-up" mass spectrometric approach.

Strader MB, Verberkmoes NC, Tabb DL, Connelly HM, Barton JW, Bruce BD, Pelletier DA, Davison BH, Hettich RL, Larimer FW, Hurst GB
J Proteome Res. 2004 3 (5): 965-78

PMID: 15473684 · DOI:10.1021/pr049940z

We present a comprehensive mass spectrometric approach that integrates intact protein molecular mass measurement ("top-down") and proteolytic fragment identification ("bottom-up") to characterize the 70S ribosome from Rhodopseudomonas palustris. Forty-two intact protein identifications were obtained by the top-down approach and 53 out of the 54 orthologs to Escherichia coli ribosomal proteins were identified from bottom-up analysis. This integrated approach simplified the assignment of post-translational modifications by increasing the confidence of identifications, distinguishing between isoforms, and identifying the amino acid positions at which particular post-translational modifications occurred. Our combined mass spectrometry data also allowed us to check and validate the gene annotations for three ribosomal proteins predicted to possess extended C-termini. In particular, we identified a highly repetitive C-terminal "alanine tail" on L25. This type of low complexity sequence, common to eukaryotic proteins, has previously not been reported in prokaryotic proteins. To our knowledge, this is the most comprehensive protein complex analysis to date that integrates two MS techniques.

MeSH Terms (22)

Acetylation Amino Acid Sequence Bacterial Proteins Chromatography, High Pressure Liquid Chromatography, Liquid Databases, Protein Escherichia coli Proteins Fourier Analysis Mass Spectrometry Methionine Methylation Molecular Sequence Data Protein Processing, Post-Translational Proteomics Rhodopseudomonas Ribosomal Proteins Ribosomes Sequence Alignment Sequence Homology Sequence Homology, Amino Acid Spectrometry, Mass, Electrospray Ionization Trypsin

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