Mass spectrometry has a key role in the Center for Molecular and Cellular Systems identifying the proteins making up the protein complexes, or "machines of life," that are the focus of the Center. Mass spectrometry is a diverse family of instrumental techniques that have in common the measurement of molecular mass, and the study of reactions gas-phase ions. For a broader view of the capabilities and applications of mass spectrometry, visit the American Society for Mass Spectrometry web site.
Several distinct types of mass spectrometric measurements are used to identify proteins in the Center for Molecular and Cellular Systems. The goal of these measurements is the identification of proteins based on measurement of (a) molecular masses of proteins or proteolytic peptides, and (b) fragmentation patterns of proteins or proteolytic peptides obtained via tandem mass spectrometry. Mass spectrometric measurements of intact proteins are called "top-down," while measurements of proteolytic peptides are called "bottom-up." All of these measurements can be performed on either "workhorse" instruments or higher-performance mass spectrometers, depending on the desired accuracy and throughput.
"Bottom-up" tandem mass spectrometric analysis of the peptides resulting from proteolytic digests of proteins is a very high throughput method for identifying proteins. In the CMCS, we use quadrupole ion trap mass spectrometry to do these analyses. The tandem mass spectrum of a peptide provides partial amino acid sequence information about the peptide. Using a combination of commercial packages and software developed collaboratively in-house, this partial sequence information can be compared with known amino acid sequences in protein databases to yield protein identifications. To accommodate the very complex mixtures of peptides resulting from proteolytic digestion of a protein complex, each of the mass spectrometers is interfaced with high-performance liquid chromatography equipment to perform on-line separations. A versatile array of single- and multi-dimensional chromatographic separations, combined with tailored mass spectometry methods, allows us to accommodate proteolytic digests ranging in complexity from simple protein mixtures (e.g. affinity-isolated protein complexes) through full microbial cell proteomes.
The quadrupole ion trap mass spectrometers used for high-throughput protein identification are robust, but do not offer particularly high performance with regard to mass measurement accuracy and resolution. Higher-performance mass spectrometers, such as Fourier transform ion cyclotron resonance or hybrid quadrupole-time-of-flight mass spectrometers, offer more powerful measurements, at the expense of speed. At PNNL, an approach called the accurate mass and time (AMT) tag method has been developed. In this approach, measurements of peptide masses are obtained that are sufficiently accurate (in the parts-per-million range) to identify the peptides without the need for tandem mass spectrometry. At ORNL, "top-down" measurements of intact protein masses are performed using Fourier transform ion cyclotron resonance mass spectrometry. These measurements allow identification of modified versions of proteins that could be missed in a "bottom-up" approach that looks only at peptides.
- Abstract: Dale A. Pelletier, et al., Protein-Protein Interactions in Rhodopseudomonas palustris at the Genomics:GTL Center for Molecular and Cellular Systems, 2009 Genomics:GTL Awardee Workshop VII, Bethesda, Maryland.
- Article: D.A. Pelletier, G.B. Hurst, L.J. Foote, P.K. Lankford, C.K. McKeown, T.-Y. Lu, D.D. Schmoyer, M.B. Shah, W.J. Hervey IV, W.H. McDonald, B.S. Hooker, W.R. Cannon, D.S. Daly, J.M. Gilmore, H.S. Wiley, D.L. Auberry, Y. Wang, F.W. Larimer, S.J. Kennel, M.J. Doktycz, J.L. Morrell-Falvey, E.T. Owens, M.V. Buchanan, "A General System for Studying Protein-Protein Interactions in Gram-Negative Bacteria," J. Proteome Research 7, 3319–3328, 2008. [PDF]
- Abstract: Michael S. Allen, et al., Characterization of a Stress Response Pathway in the Anoxygenic Phototrophic Bacterium Rhodopseudomonas palustris, 2008 Genomics:GTL Awardee Workshop VI, Bethesda, Maryland.
- Abstract: Dale A. Pelletier, et al., Protein-Protein Interactions Involved in Electron Transfer to Nitrogenase for Hydrogen Production in Rhodopseudomonas palustris, 2008 Genomics:GTL Awardee Workshop VI, Bethesda, Maryland.
- Abstract: Kevin K. Anderson, et al., Advanced Data Analysis Pipeline for Determination of Protein Complexes and Interaction Networks at the Genomics:GTL Center for Molecular and Cellular Systems, 2007 Genomics:GTL Awardee Workshop V, Bethesda, Maryland.
- Article: R.J. Giannone, W.H. McDonald, G.B. Hurst, Y. Huang, Y. Liu, Y. Wang, "A Dual-Tagging System for Affinity Purification of Mammalian Protein Complexes," BioTechniques 2007, 43, 296-302. [PDF]
- Article: W.J. Hervey, IV, M.B. Strader, and G.B. Hurst, "Comparison of Digestion Protocols for Microgram Quantities of Enriched Protein Samples," J. Proteome Research, 6, 3054-3061, 2007. [PDF]
- Presentation: Gregory B. Hurst, Dale A. Pelletier, Stephen J. Kennel, Frank W. Larimer, W. Hayes McDonald, Trish K. Lankford, Manesh B. Shah, Denise D. Schmoyer, Tse-Yuan S. Lu, Linda J. Foote, Cathy K. McKeown, Jenny L. Morrell-Falvey, Mitchel J. Doktycz, and Michelle V. Buchanan, "High Throughput Identification of Protein Complexes from Rhodopseudomonas palustris," Oral presentation at LabAutomation 2007, Palm Springs, California, January 29, 2007.
- Presentation: B.S. Hooker, D.L. Auberry, K.J. Auberry, W.R. Cannon, Y.A. Gorby, E.A. Hill, V. Kery, C.T. Lin, E.A. Livesay, P.A. Moore, R.J. Moore, R.D. Smith, K.D. Victry, H.S. Wiley, D. Pelletier, M. Buchanan, L. Foote, G. Hurst, S. Kennel, T. Lankford, T. Lu, W. McDonald, C. McKeown, and D. Schmoyer. "Determination of Protein Interaction Subnetworks in Shewanella oneidensis Using Endogenous and Exogenous Affinity Isolation Methods," Presented at the 2007 Society for Biological Engineers International Conference on Biomolecular Engineering, San Diego, California, January 2007.
- Presentation: Hayes McDonald, "Beyond model systems - protein complexes," Oral presentation at Cambridge Health Institute Pep Talk Conference, San Diego, California, January 12, 2007.
- Abstract: Jennifer Morrell-Falvey, et al., Advances in Coverage and Quality for High-Throughput Protein-Protein Interaction Measurements, 2007 Genomics:GTL Awardee Workshop V, Bethesda, Maryland .
- Abstract: Dale A. Pelletier, et al., Global Survey of Protein-Protein Interactions in Rhodopseudomonas palustris, 2007 Genomics:GTL Awardee Workshop V, Bethesda, Maryland.
- Article: J.L. Sharp, K.K. Anderson, G.B. Hurst, D.S. Daly, D.A. Pelletier, W.R. Cannon, D.L. Auberry, D.D. Schmoyer, W.H. McDonald, A.M. White, B.S. Hooker, K.D. Victry, M.V. Buchanan, V.Kery, and H.S. Wiley, "Statistically Inferring Protein-Protein Associations with Affinity Isolation LC-MS/MS Assays," J. Proteome Research, 6, 3788-3795, 2007. [PDF]
- Abstract: H.M. Connelly, D.A. Pelletier, T-Y. Lu, P.K. Lankford, and R.L. Hettich, "Characterization of pII Family (GlnK1, GlnK2, GlnB) Protein Uridylylation in Response to Nitrogen Availability for Rhodopseudomonas palustris," Anal. Biochem. 2006, 357, 93-104.
- Abstract: R. Hettich, et al., Advanced Technologies for Identifying Protein-Protein Interactions, 2006 Contractor-Grantee Workshop, North Bethesda, MD (PDF)
- Abstract: Chiann-Tso Lin, Priscilla A. Moore, Deanna L. Auberry, Elizabeth V. Landorf,Terese Peppler, Kristin D. Victry, Frank R. Collart, and Vladimir Kery, "Automated purification of recombinant proteins: Combining high-throughput with high yield," Protein Expression and Purification 47, 16–24, 2006. [PDF]
- Abstract: D. Pelletier, et al., Comparison of Conserved Protein Complexes Across Multiple Microbial Species to Evaluate High-Throughput Approaches for Mapping the Microbial Interactome, 2006 Contractor-Grantee Workshop, North Bethesda, MD (PDF)
- Article: Nathan C. VerBerkmoes, Manesh B. Shah, Patricia K. Lankford, Dale A. Pelletier, Michael B. Strader, David L. Tabb, W. Hayes McDonald, John W. Barton, Gregory B. Hurst, Loren Hauser, Brian H. Davison, J. Thomas Beatty, Caroline S. Harwood, F. Robert Tabita, Robert L. Hettich, and Frank W. Larimer, "Determination and Comparison of the Baseline Proteomes of the Versatile Microbe Rhodopseudomonas palustris under Its Major Metabolic States ," J. Proteome Res. 5, 287-98, 2006
- Article: William R. Cannon, Kristin H. Jarman, Bobbie-Jo M. Webb-Robertson, Douglas J. Baxter, Christopher S. Oehmen, Kenneth D. Jarman, Alejandro Heredia-Langner, Kenneth J. Auberry, and Gordon A. Anderson, "Comparison of Probability and Likelihood Models for Peptide Identification from Tandem Mass Spectrometry Data," Journal of Proteome Research, 4(5), 1687-1698, 2005. [PDF]
- Article: R. S. Foote, J. Khandurina, S. C. Jacobson, and J. M. Ramsey, "Preconcentration of proteins on microfluidic devices using porous silica membranes", Anal. Chem., 77 (1), 57-63, 2005.
- Abstract: Sara P. Gaucher, Masood Z. Hadi, and Malin M. Young, Investigating Gas Phase Dissociation Pathways of Crosslinked Peptides: Application to Protein Complex Determination, 2005 Genomics:GTL Contractor-Grantee Workshop, Washington, DC.
- Abstract: Vladimir Kery, et al., High-Throughput Analysis of Protein Complexes in the Center for Molecular and Cellular Systems, 2005 Genomics:GTL Contractor-Grantee Workshop, Washington, DC.
- Article: C.T. Lin, L.M. Markillie, T.C. Squier, B.S. Hooker, L. Shi, "An improved system for functional over-expression of multi-heme c-type cytochromes," Biotechniques 38, 297-99, 2005.
- Article: L. M. Markillie, C.-T. Lin, J. N. Adkins, D. L. Auberry, E. A. Hill, B. S. Hooker, P. A. Moore, R. J. Moore, L. Shi, H. S. Wiley, V. Kery, "Simple Protein Complex Purification and Identification Method for High-Throughput Mapping of Protein Interaction Networks," J. Proteome Res., 2005, 4(2); 268-274. DOI: 10.1021/pr049847a [PDF]
- Article: Jane M. Weaver-Feldhaus, Keith D. Miller, Michael J. Feldhaus, and Robert W. Siegel, "Directed evolution for the development of conformation-specific affinity reagents using yeast display," Protein Engineering, Design & Selection 18(11): 527–536, 2005 . [PDF]
- Abstract: Michelle V. Buchanan, et al., Establishment of Protocols for the High Throughput Analysis of Protein Complexes at the Center for Molecular and Cellular Systems, 2004 Genomics:GTL Contractor-Grantee Workshop, Washington, DC .
- Abstract: Brian S. Hooker, et al., Isolation and Characterization of Protein Complexes from Shewanella oneidensis and Rhodopseudomonas palustris, 2004 Genomics:GTL Contractor-Grantee Workshop, Washington, DC .
- Abstract: Dale A. Pelletier, et al., High-Throughput Cloning, Expression and Purification of Rhodopseudomonas palustris and Shewanella oneidensis Affinity Tagged Fusion Proteins for Protein Complex Isolation, 2004 Genomics:GTL Contractor-Grantee Workshop, Washington, DC
- Article: J. Weaver-Feldhaus, J. L. Lou, J. R. Coleman, R. W. Siegel, J. D. Marks, M. Feldhaus, 2004. "Yeast mating for combinatorial Fab library generation and surface display." FEBS Letters. 564:24-34.
- Presentation: "Cross-Linking," 2003 Genomes to Life Contractor-Grantee Workshop, Arlington, Virginia [Web or PDF]
- Abstract: Michelle Buchanan, et al., New Approaches for High-Throughput Identification and Characterization of Protein Complexes, 2003 Genomes to Life Contractor-Grantee Workshop, Arlington, Virginia
- Images and captions: Michael Giddings and the University of North Carolina, 2003 Genomes to Life Contractor-Grantee Workshop, Arlington, Virginia [PDF]
- Abstract: P. R. Hoyt, et al., Automation of Protein Complex Analyses in Rhodopseudomonas palustris and Shewanella oneidensis, 2003 Genomes to Life Contractor-Grantee Workshop, Arlington, Virginia
- Poster: Richard D. Smith, et al., "PNNL Protein Complex Characterization Efforts," 2003 Genomes to Life Contractor-Grantee Workshop, Arlington, Virginia [PDF shorter download or higher quality]
- Abstract: Hurst, et al., Investigating Protein Complexes by Crosslinking and Mass Spectrometry, 2002 DOE Genome Contractor-Grantee Workshop, Oakland, CA