# Presentation: 716589

## Analysis of Protein Complexes from Rhodopseudomonas palustris by Mass SpectrometryGregory B. Hurst, Dale A. Pelletier, Robert L. Hettich, Keiji G. Asano, Michael B. Strader, David L. Tabb, Nathan C. VerBerkmoes, Trish K. Lankford, Linda J. Foote, Yisong Wang, Stephen J. Kennel, Frank W. LarimerOak Ridge National Laboratory, Oak Ridge, TN

- OVERVIEW

- Analysis of Protein Complexes from *Rhodopseudomonas palustris *by Mass SpectrometryGregory B. Hurst, Dale A. Pelletier, Robert L. Hettich, Keiji G. Asano, Michael B. Strader, David L. Tabb, Nathan C. VerBerkmoes, Trish K. Lankford, Linda J. Foote, Yisong Wang, Stephen J. Kennel, Frank W. LarimerOak Ridge National Laboratory, Oak Ridge, TN

- INTRODUCTION

**Target proteins in *****Rhodopseudomonas palustris***** have been expressed as fusions with affinity labels to enable isolation of protein complexes.**
**Component proteins of several complexes have been affinity-isolated and characterized by MS.**

- The bacterial species *Rhodopseudomonas palustris* [1]
- occurs widely in the environment
- survives in a variety of conditions
- light / dark
- aerobic / anaerobic
- This species thus has the potential to express markedly different complements of proteins and protein complexes under different growth conditions.
- As part of a center funded by the U.S. Department of Energy Genomes To Life program [2,3], we are analyzing protein complexes from *R. palustris* by expressing target proteins as fusions with affinity tags to allow subsequent isolation of other proteins associated with the target [4].
- The MS analysis approach includes
- Bottom-up analysis of affinity-isolated complexes (this poster)
- Bottom-up proteomics of *R. palustris* proteins (poster ThPT 382, VerBerkmoes *et al.*)
- Top-down analysis of intact proteins by FT-ICR-MS (poster MPU 390, Connelly *et al.*)
- Integrated top-down and bottom-up characterization [5] of a conventionally-isolated complex (70S ribosome from *R. palustris*). [6] (poster ThPN 270, Strader *et al.*)

- REFERENCES

- This research sponsored by the Office of Biological and Environmental Research, U.S. Department of Energy.  Oak Ridge National Laboratory is managed by UT-Battelle, LLC, for the U.S. Department of Energy under Contract No. DE-AC05-00OR22725.

- 1. 	Larimer, F. W. *et al.*, “Complete genome sequence of the metabolically versatile photosynthetic bacterium *Rhodopseudomonas palustris*.” *Nature Biotech.*** 2004**, *22*, 55-61.
- 2. 	http://www.genomestolife.org
- 3. 	Buchanan, M. V. et al., “Genomes to Life ‘Center for Molecular and Cellular Systems:’  research program for identification and characterization of protein complexes.” *OMICS* **2002**, *6*, 287-303.
- 4. 	(a) Gavin, A.-C. et al., "Functional organization of the yeast proteome by systematic analysis of protein complexes," *Nature ***2002**, *415, *141-147.
-    	(b) Ho, Y. *et al*., "Systematic identification of protein complexes in *Saccharomyces cerevisiae* by mass spectrometry," *Nature ***2002**, *415, *180-183.
- 5.  	Verberkmoes, N.C. et al., “Integrating “top-down” and “bottom-up” mass spectrometric approaches for proteomic analysis of *Shewanella oneidensis*,” *J. Proteome Res.* **2002**, *1*, 239-252.
- 6.  	Strader, M.B., *et al.*, “Analysis of the 70S ribosome from *Rhodopseudomonas palustris* using integrated top-down and bottom-up mass spectrometric approaches.”   Sixth International Symposium on Mass Spectrometry in the Health and Life Sciences:  Molecular and Cellular Proteomics, San Francisco, CA, Aug. 24-28, 2003.
- 7.  	Puig, O. *et al*.,  "The tandem affinity purification (TAP) method: a general procedure of protein complex purification," *Methods ***2001**, *24, *218-229.
- 8.  	Link, A.J. *et al*.,  "Direct analysis of protein complexes using mass spectrometry," *Nature Biotech. ***1999**, *17, *676-682.
- 9. 	Eng, J. K. *et al.*, “An approach to correlate tandem mass spectral data of peptides with amino acid sequences in a protein database.” *J. Am. Soc. Mass Spectrom.* **1994**, *5*, 976-89.
- 10.	Russell, W. K. *et al.*,  “Proteolysis in Mixed Organic-Aqueous Solvent Systems: Applications for Peptide Mass Mapping Using Mass Spectrometry.” *Anal. Chem.* **2001, ***73*, 2682-85.

**PCR amplification of target gene and cloning **
**into modified expression vector**

**Transfer expression plasmid **
**into *****R. palustris***

**Expression of affinity tagged proteins **
**under various physiological conditions**

**Capture protein complexes**
**using affinity tag**

**ID proteins**

**Stoichiometries**

**Functional assays**

**Characterize Protein Complex**

**Mass Spectrometry**

- Selected *R. palustris* genes were cloned with affinity tags [7] and expressed in both *E. coli* and *R. palustris* using modified pDEST vectors (Invitrogen).
- Tag contains both His6 and V5 epitope
- C-terminal position
- Isolation of fusion proteins
- Affinity purification with Ni-NTA agarose beads, followed by anti-V5 antibody agarose beads
- Expression confirmed using 1-D PAGE and western blots.
- “Shotgun” analysis: analysis by mass spectrometry without prior gel separation [8]
- trypsin digestion
- Reverse-phase HPLC separation online with electrospray/quadrupole ion trap MS-MS
- Protein ID’s: Sequest [9] analysis of tandem mass spectral data using *R. palustris* database [1] .

- RESULTS

- Plasmids encoding 42 affinity-tagged fusion proteins have been inserted into *R. palustris* (**Table 1**).
- Choice of target proteins guided by proteomics measurements (poster ThPT 382, VerBerkmoes *et al.*)
- Multiple protein components of several complexes have been identified:
- Iron nitrogenase (**Table 2**)
- RNA polymerase (**Table 3**)
- ATP synthase
- GroESL
- Over 400 “background” (i.e., unlabeled) proteins were commonly observed in numerous LC-MS-MS experiments. **Figure 1 **shows the most frequently detected of these.
- Based on this expanding library of background measurements, we are currently developing criteria for determining whether detection of a given protein in a particular isolation/mass spectrometry experiment represents a valid “hit.”
- One rough indication of a valid “hit” may be whether selected parameters for a detection (e.g. sequence coverage, number of unique peptides, total number of peptide spectra) significantly exceed those observed in the background for that protein.
- As more measurements are made, the background library expands, and “hits” can be continually re-evaluated and refined.

**Significant progress is reported in expression, isolation, and detection of protein complexes in *****R. palustris*****.  **
**Development of more robust methods for identifying proteins associated with the tagged protein is underway, based on the expanding, well-characterized background library that we are amassing.**
**Future plans include:**
**Implementation of improved separations techniques, online with mass spectrometry, that provide wider dynamic range and potentially faster throughput**
**Integration with high-mass-accuracy measurements of both peptides and intact proteins.**
**Development of digestion protocols that are faster, amenable to smaller sample sizes, and more compatible with subsequent MS analysis. [10]**

**Table 2:  Isolation of Nitrogenase complex**
- Three proteins in a nitrogenase complex (nifD, nifH, nifK) were each cloned with both His6 and V5 epitope tags, expressed under photoheterotrophic, nitrogen-fixing conditions, and isolated.
- LC-MS-MS analysis of the isolate from each labeled component showed evidence for all three components of the nitrogenase complex.

| Tagged
Protein: | nifK |  | nifD |  | nifH |  |
| --- | --- | --- | --- | --- | --- | --- |
|  | seq
cov. (%) | unique
peptides | seq
cov. (%) | unique
peptides | seq
cov. (%) | unique
peptides |
| nifK | 47 | 19 | 54 | 31 |   6 | 2 |
| nifD | 41 | 19 | 63 | 38 | 14 | 4 |
| nifH | 22 |   4 | 17 |   3 | 29 | 9 |

**Table 3:  Isolation of RNA Polymerase complex**
- The alpha subunit of a DNA-directed RNA polymerase complex was cloned with both His6 and V5 epitope tags, expressed in *R. palustris*, and isolated.
- LC-MS-MS analysis of the isolate showed evidence for the beta, beta’, and omega subunits of the complex.  Additionally, two sigma factors for this polymerase were observed.  These sigma factors are sub-stoichiometric, and associate with the polymerase under different conditions of growth or stress.

| Gene | Description | unique
peptides | sequence
coverage (%) |
| --- | --- | --- | --- |
| RPA3226 (tagged) | DNA-directed RNA polymerase alpha subunit | 37 | 73 |
| RPA2692 | RNA polyerase omega subunit |   4 | 53 |
| RPA3268 | RNA polymerase beta subunit | 50 | 42 |
| RPA3267 | RNA polyerase beta’ subunit | 28 | 26 |
| RPA1288 | RNA polymerase sigma subunit |   3 | 11 |
| RPA0367 | Stress response sigma factor |   2 |   6 |

- CONCLUSIONS

- ACKNOWLEDGEMENTS

- EXPERIMENTAL