Protein Characterization

Protein Characterization

The Proteomics Centre offers a number of services and unique strategies for characterizing proteins based on mass spectrometric methods and approaches developed at the Centre and through Dr. Borchers’ research, including bottom-up and top-down proteomic approaches. Bottom-up approaches enable the analysis of peptides obtained by proteolysis and are particularly useful for the characterization of specific post-translational modifications including phosphorylation, acetylation, ubiquitination, and glycosylation. Top-down techniques allow for the direct analysis of intact proteins. The Centre has developed and applied such approaches to determine the composition of protein complexes, to accurately reveal the truncation site of proteins, and to elucidate the distribution and types of protein isoforms. Detailed information of the bottom-up and top-down proteomic approaches are described in Dr. Borchers’ publications.

Bottom-up Approaches:

• Phosphorylation

  1. Domanski, D., Murphy, L., Borchers, C. Assay Development for the Determination of Phosphorylation Stoichiometry using MRM methods with and without Phosphatase Treatment: Application to Breast Cancer Signaling Pathways. Anal Chem, 82(13): 5610-20. (2010)
  2. Torres, M.P., Borchers, C.H., Phosphorylation of the APC Inhibitory Subunit Mnd2 is Necessary for Efficient Progression Through Meiosis. JBC, 282(24): 62. (2007)
  3. Borchers, C.H., Thapar, R., Petrotchenko, E.V., Torres M.P., Speir, S.P., Easterling M., Dominski, Z., Marzluff, W.F., Combined Top-down and Bottom-up Proteomics Identifies a Phosphorylation Site in Stem-Loop Binding Proteins That Contributes to High-affinity RNA Binding. PNAS, 103(9): 3094-3099.(2006)
  4. Torres, P.T., Thapar, R., Marzluff, W.F., Borchers, C.H., Phosphatase-Directed Phosphorylation-Site Determination: A synthesis of Methods for the Detection and Identification of Phosphopeptides. J Prot Res, 4(5): 992-7. (2005)
  5. Parnell, S.C., Marotti, L.A., Kiang, L., Torres, M.P., Borchers, C.H., Dohlman, H.G., Phosphorylation of the RGS Protein Sst2 by the MAP kinase Fus3 and use of Sst2 as a Model to Analyze Determninats of Substrate Specificity. Biochemistry. 44(22): 8159-66. (2005)
  6. Hall, M.C., Warren, E.N., Borchers C.H.,Multi-Kinase Phosphorylation of the APC/C Activator Cdh1 Revealed by Mass Spectrometry. Cell Cycle. 3: 1278-1284. (2004)
  7. Huang, C., Borchers, C.H., Schaller, M.D., Jacobson, K., Phosphorylation of paxillin by p38mapk is involved in the neurite outgrowth of PC-12 cells. J Cell Biol, 164(4): 593-602. (2004)
  8. Xiao, T., Hall, H., Kizer, K., Sabata, Y., Hall, M., Borchers, C.H., Strahl, B.D., Phosphorylation of RNA polymerase II CTD regulates H3 methylation in yeast, Genes & Dev. 17: 654-663. (2003)
  9. Huang, C., Rajfur, Z., Schaller, M., Borchers C., Jacobson K., Phosphorylation of Serine 178 on Paxillin by JNK1 can regulate Cell Migration. Nature, 424(6945): 219-223. (2003)
  10. Zheng, L., Dominski, Z., Yang, X-C., Elms, P., Raska, C., Borchers, C.H., Marzluff, W.F., Phosphorylation of SLBP on two threonines triggers degradation of SLBP, the sole cell-cycle regulated factor required for regulation of histone mRNA processing, at the end of S-phase, Mol Cell Biol 23(5): 1590-1601.(2003)
  11. Jun, H., Pope, R.M., Borchers, C., Graves, L.M., Mapping of Protein Phosphorylation by Dual Enzyme Digestion on MALDI-QoTOF-MS. Anal Biochem, 310(2): 214-218. (2002)
  12. Dominski, Z., Yang, X., Raska, C.S., Santiago, C., Borchers, C.H., Duronio, R.J., Marzluff, W.F., 3'end processing of Drosophila histone pre-mRNAs: Requirement for phosphorylated dSLBP and co-evolution of the histone pre-mRNA processing system. Mol Cell Biol, 22(18): 6648-6660. (2002)
  13. Raska, C.S., Parker, C.E., Glish, G.L., Dominski, Z., Marzluff, W.F., Pope, R.M., Borchers, C.H., Direct MALDI-MS/MS of Phosphopeptides Affinity-Bound to IMAC Beads. Anal Chem, 74: 3429-3433. (2002)
  14. Merrick, B.A., Zhou, W., Martin, K.J., Jeyarajah, S., Parker,C.E., Selkirk, J.K., Tomer, K.B., Borchers, C.H., Site-Specific Phosphorylation of Human P53 Protein Determined by Mass Spectrometry. Biochemistry, 39: 11194-11204. (2001)

• Acetylation

  1. Borchers, C., Parker, C.E., Deterding, L.J., Tomer, K.B., A Preliminary Comparison of Precursor Scans and LC/MS/MS on a Hybrid Quadrupole Time-of-flight Mass Spectrometry. J. Chromatogr. A. (1999) 854: 119-130.

• Ubiquitination

  1. Warren, M.R.E., Parker, C.E., Mocanu, V., Klapper, D., Borchers C.H., Electrospray Ionization Tandem Mass Spectrometry of Model Peptides Reveals Diagnostic Fragment Ions for Protein Ubiquitination. Rap. Commun. Mass Spectrom. (2005) 19: 1-9.
  2. Parker, C.E., Warren, M.R., Mocanu, V., Greer, S.F.,Borchers, C.H., Mass spectrometric determination of protein ubiquitination. Methods Mol Biol. (2008) 446, 109-30.

• Glycosylation

  1. Xuegong, Z., Borchers, C., Bienstock, R.J., Tomer, K.B., Mass Spectrometric Characterization of the Glycosylation Pattern of HIV-gp120 Expressed in CHO Cells. Biochemistry. (2000) 39: 11194-11204.

Top-down approaches:

• Truncation

  1. McMorran B.J., Ouvry Patat S.A., Carlin J.B, Grimwood K., Jones A., Armstrong D.S., Galati J.C., Cooper P.J., Byrnes C.A., Francis P.W., Robertson C.F., Hume D.A., Borchers C.H., Wainwright C.E., Wainwright B.J. Proteomic Profiling reveals novel markers of the exaggerated pulmonary inflammatory response in paediatric cystic fibrosis bronchoalveolar lavage fluid. Journal of Clinical Chemistry. (2007) 53(10): 1782 - 91.
  2. Hall, M.C., Shcherbakova, P.V., Borchers, C.H., Dial, J.M., Tomer, K.B., Kunkel, T.A., DNA binding by yeast Mlh1 and Pms1: implications for DNA mismatch repair. Nucleic Acids Research. (2003) 31(8): 2025-2034.
  3. Sinclair, C., Borchers, C., Parker, C., Tomer, K., Charbonneau, H., Rossie, S., The Tetratricopeptide Repeat Domain and a C-terminal Region Control the Activity of Ser/Thr Protein Phosphatase 5. J. Biol. Chem.(1999) 274: 23666-23672.

• Posttranslational Modifications

  1. Han, J. and Borchers, C.H.Top-down analysis of recombinant histone H3 and its methylated analogs by ESI/FTICR mass spectrometry. Proteomics, (2010)
  2. Sneekes, E., Han, J., Elliot, M., Ausio, J., Swart, J., Heck, A.J. and Borchers, C.H. Accurate molecular weight analysis of histones using FFE and RP-HPLC on monolithic capillary columns. J Sep Sci, 32(15-16): 2691-8. (2009)
  3. Ouvry-Patat, S.A., Torres, M.P., Gelfland, G., Easterling, M., Speir, J.P. and Borchers, C.H. Top-down proteomics on a high-field Fourier transform ion cyclotron resonance mass spectrometer. Meth Mol Biol, 492:215-31 (2009)
  4. Ouvry-Patat, S.A., Torres, M.P., Quek, H., Gelfand, C.A., Schroeder, G.K., Han, J., Elliott, M., Dryhurst, D., Ausio, J., Wolfenden, R., Borchers, C.H., Free Flow Electrophoresis for Top-Down Proteomics by Fourier Transform Ion Cyclotron Resonance Mass Spectrometry. Proteomics. (2008) 8:2798-2808.
  5. Borchers, C.H., Thapar, R., Petrotchenko, E.V., Torres M.P., Speir, S.P., Easterling M., Dominski, Z., Marzluff, W.F., Combined Top-down and Bottom-up Proteomics Identifies a Phosphorylation Site in Stem-Loop Binding Proteins That Contributes to High-affinity RNA Binding. PNAS. (2006) 103(9): 3094-3099.

• Protein Complex Composition

  1. Petrotchenko, E.V., Serpa J., and Borchers, C.H. An isotopically-coded CID-cleavable biotinylated crosslinker for structural proteomics. Mol Cell Proteomics. (2010)
  2. Petrotchenko, E.V. and Borchers, C.H. Crosslinking combined with mass spectrometry for structural proteomics. Mass Spec Rev. (2010)
  3. Pan J., Han, J., Borchers, C.H., Konermann, L. Hydrogen/Deuterium Exchange Mass Spectrometry with Top-Down Electron Capture Dissociation for Characterizing Structural Transitions of a 17 kDa Protein. J Am Chem Soc. 131(35) :12801-8. (2009)
  4. Laith, Q.A-M., Fikkert, V., Dayam, R., Burke, T.R., Borchers, C.H., Neamti, N. Discovery of a novel small molecule HIV-1 integrase inhibitor binding-site through photoaffinity labeling and mass spectrometry. PNAS. (2006) 103(26): 10080-10085.
  5. Borchers, C.H., Marquez, V.E., Schroeder, G.K., Short, S.A., Snider, M.J., Speir, P.K., Wolfenden, R., Fourier Transform Ion Cyclotron Resonance MS Reveals the Presence of a Water Molecule in an Enzyme Transition-State Analogue Complex. PNAS. (2004) 101(43): 15341-15345.