Characterization of the 70S Ribosome from Rhodopseudomonas palustris Using an Integrated TopDown and

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Characterization of the 70S Ribosome from
Rhodopseudomonas palustrisUsing an Integrated
Top-Down and Bottom-Up Mass Spectrometric
Approach

• Authors 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

Presented by Kathy Goodson
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Overview

• Top-Down and Bottom-Up
• LC-MS-MS
• SEQUEST
• TDBU Analysis
• Post-translational Modifications
• Isoforms
• R. palustris Ribosomal Proteins
• Conclusions

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Top-Down and Bottom-Up
Characterization of the 70S Ribosome from
Rhodopseudomonas palustris
strategies for identifying multiprotein complexes
on a genome-wide scale

• mass spectrometric approach that incorporates
  optimal identification

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Top-Down and Bottom-Up

• Top-Down
• Approach that involves starting with the intact
  protein mass
• Bottom-Up
• Approach that involves starting with proteolytic
  fragments to piece the protein back together

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Top-Down and Bottom-Up
Top-Down

• Cons
• Lack of bioinformatics tools for data analysis
  and verification of PTMs

• Pros
• Potential identification of translational start
  and stop sites, mRNA splice variants, and
  Post-translational modifications (PTMs)

Bottom Up

• Cons
• PTMs may be overlooked
• Involves peptide analysis from a complex mixture

• Pros
• Identifying large numbers of proteins in a single
  data acquisition

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Integration of Top-Down and Bottom-Up

• Assignment of post-translational modifications
  and the amino acid position where they take place
• Distinction between isoforms
• Overall the increased confidence of such
  identifications

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Integration of Top-Down and Bottom-Up
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LC-MS-MS for Bottom-Up Analysis
One-dimensional (1D) separations
Two-dimensional (2D) separations
Data dependent MS-MS mode
Four parent ions from each mass spectrum were
chosen based on abundance and absence from
exclusion list
Performed over a parent ion m/z range of
400-2000. In addition, separate injections were
made while scanning several narrower parent ion
ranges (m/z 400-1000, m/z 980-1500, and m/z
1480-2000)
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Bottom-Up Analysis

• Initial search included only tryptic peptides
  from R. palustris proteins with no PTMs
• Preliminary results were then reanalyzed using
  SEQUEST by searching against
• all predicted peptides
• Spectra from singly charged peptides were
  required to exceed 1.8 in XCorr

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SEQUEST
Peptide from protein digest sequenced by MS/MS

• Identification of the AA sequence in the database
  correlating to the measured mass of the selected
  peptide
• Prediction of the fragmentation pattern that is
  expected for each sequence
• The fragmentation patterns are matched with the
  experimentally derived MS/MS spectrum

Report of high scoring AA sequences
C. Dass (2001) Principles and Practice of
Biological Mass Spectrometry, John Wiley Sons,
Inc., New York, pp. 194-195.
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Bottom-Up Analysis

• Four-mass-range 1D and the 2D
• strategies resulted in similar
• identification and overall
• sequence coverage from the
• SEQUEST searches

• In general, 1D and 2D separations
• where found to be complementary in
• regard to quality of results obtained,
• amount of sample required, and time
• requirements

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Top-Down Analysis
Ion detection was achieved in an ultrahigh
vacuum regime (2 x 10-10 Torr)
Mass resolution of at least 50,000 at m/z 1000
Deconvoluted molecular mass spectra generation
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Top Down

• Most abundant
• isotopic mass
• (MAIM) peak was
• used to query the
• entire R. palustris
• protein database

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TDBU

• Truncation of the start methionine was the most
  common identified PTM
• The top-down technique identified an N-terminal
  truncation if the measured intact mass for a
  protein matched that obtained by subtracting the
  methionine residue mass
• The bottom-up technique identified N-terminal
  peptides without a methionine

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Post-translational Modifications

• One of the bottom-up data sets agreed with top
  down data

• Or if bottom-up data from both the 1D and 2D
  separations were
• consistent

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Identification of Isoforms

• MS-MS spectra
• representing peptides with
• di-methylated K69 and
• mono-methylated
• K86 residues

• The top-down
• molecular mass of
• 12,754.089 Da indicated
• that this protein was
• modified by methionine
• truncation, plus either
• multiple methylation or
• acetylation.

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R. palustris Ribosomal Proteins

• The DNA derived sequence for RRP-L25 predicted an
  unusual extended C-terminus deemed a
  poly-alanine tail
• The only other bacterium found to possess an L25
  protein with this C-terminal poly-alanine tail
  was B. japonicum.
• Has not previously been identified at the protein
  level in prokaryotes.
• Bottom-up data from both 1D and 2D LC separations
  included peptides for the entire C-terminal
  extension (with the exception of the last three
  lysine residues).

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Conclusions

• 53 of 54 E. coli ribosomal proteins were
  identified from bottom-up MS analysis
• 42 intact protein identifications were obtained
  by the top-down approach
• Three ribosomal proteins with unusual extended
  sequences