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Article

Characterization of the 70S Ribosome from Rhodopseudomonas palustris Using an Integrated “Top-Down” and “Bottom-Up” Mass Spectrometric Approach

Supporting Info

Michael Brad Strader,^† Nathan C. VerBerkmoes,^†^‡ David L. Tabb,^§ Heather M. Connelly,^†^‡ John W. Barton,^§ Barry D. Bruce, Dale A. Pelletier,^§ Brian H. Davison,^§ Robert L. Hettich,^†^‡ Frank W. Larimer,^§ and Gregory B. Hurst*^†

Organic and Biological Mass Spectrometry Group, Chemical Sciences Division, Oak Ridge National Laboratory, P.O. Box 2008, Oak Ridge, Tennessee 37831-6131, Life Sciences Division, Oak Ridge National Laboratory, Oak Ridge Tennessee 37831, Graduate School of Genome Science and Technology, University of Tennessee- Oak Ridge National Laboratory, 1060 Commerce Park, Oak Ridge, Tennessee 37830-8026, and Biochemistry Cellular and Molecular Biology Department, University of Tennessee, Knoxville, Tennessee 37996

Journal of Proteome Research, 2004, 3 (5), pp 965–978

DOI: 10.1021/pr049940z

Publication Date (Web): August 14, 2004

†

Organic and Biological Mass Spectrometry Group, Chemical Sciences Division, Oak Ridge National Laboratory.

‡

Graduate School of Genome Science and Technology, University of Tennessee-Oak Ridge National Laboratory.

Life Sciences Division, Oak Ridge National Laboratory.

Biochemistry Cellular and Molecular Biology Department, University of Tennessee.

To whom correspondence should be addressed. Phone: (865) 574−6142. Fax: (865) 576−8559. E-mail: hurstgb@ornl.gov.

Abstract

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.

Keywords: bottom-up • top-down • Rhodopseudomonas palustris • protein complexes • 70S ribosome • post-translational modifications • proteomics • mass spectrometry

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