Characterization of the 70S Ribosome from Rhodopseudomonas palustris using an integrated "top-down" and "bottom-up" mass spectrometric approach.
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.
Organic and Biological Mass Spectrometry Group, Chemical Sciences Division, Oak Ridge National Laboratory, P.O. Box 2008, Oak Ridge, Tennessee 37831-6131, USA.
Michael Brad
MB
Strader
Nathan C
NC
Verberkmoes
David L
DL
Tabb
Heather M
HM
Connelly
John W
JW
Barton
Barry D
BD
Bruce
Dale A
DA
Pelletier
Brian H
BH
Davison
Robert L
RL
Hettich
Frank W
FW
Larimer
Gregory B
GB
Hurst
D000107:Acetylation; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000032:analysis / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D002853:Chromatography, Liquid; D030562:Databases, Protein; D029968:Escherichia coli Proteins; D005583:Fourier Analysis; D013058:Mass Spectrometry / Q000379:methods; D008715:Methionine / Q000737:chemistry; D008745:Methylation; D008969:Molecular Sequence Data; D011499:Protein Processing, Post-Translational; D040901:Proteomics / Q000379:methods*; D012241:Rhodopseudomonas / Q000378:metabolism*; D000090822:Ribosomal Protein L3; D012269:Ribosomal Proteins / Q000032:analysis* / Q000737:chemistry; D012270:Ribosomes / Q000378:metabolism*; D016415:Sequence Alignment; D017385:Sequence Homology; D017386:Sequence Homology, Amino Acid; D021241:Spectrometry, Mass, Electrospray Ionization; D014357:Trypsin / Q000378:metabolism
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; C489050:L33 ribosomal protein, bacteria; D000090822:Ribosomal Protein L3; D012269:Ribosomal Proteins; C027624:ribosomal protein L11; C031127:ribosomal protein L25; C035201:ribosomal protein L30; C049650:ribosomal protein L9; C034212:ribosomal protein S12; C030004:ribosomal protein S2; C512844:rplL protein, E coli; D008715:Methionine; D014357:Trypsin