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developer of the AIDS-HIV Reference project
Abstract No.: L2
Speaker: Donald F. Hunt
Session: Plenary Lecture L2
Presentation date: Tue, Aug 29, 2006
Presentation time: 09:00 – 09:50

Comparative Analysis of Post-Translationally Modified Proteins and Peptides by Mass Spectrometry: New Technology (Electron Transfer Dissociation) and Applications in the Study of Cell Migration, the Histone Code and Cancer Vaccine Development.

Donald F. Hunt1

1 University of Virginia, Charlottesville, United States

Correspondence address: Donald F. Hunt, University of Virginia, Chemistry Department, McCormick Road, Charlottesville, VA, 22904-4319 United States.

Keywords: Immunology, Class I Peptide Antigens; MS/MS, Liquid Chromatography; Peptide, Fragmentation Mechanisms.

For the direct analysis of proteins on a chromatographic time scale, we use monolithic nanocolumns, a modified linear ion trap mass spectrometer, and sequential ion/ion reactions to fragment the intact protein and convert all fragments to singly charged species.1,2 Proteins are converted to gas-phase multiply charged positive ions by electrospray ionization and then allowed to react with fluoranthene radical anions. Electron transfer to the multiply charged protein promotes random fragmentation of amide bonds along the protein backbone. Multiply charged fragment ions are then deprototonated in a second ion/ion reaction with the carboxylated anion of benzoic acid. The m/z values for the resulting singly and doubly charged ions are used to read a sequence of 15-40 amino acids at both the N and C termini of the protein. This information, along the the measured mass of the intact protein is used to identify unknown proteins, to confirm the amino acid sequence of a known protein, to detect posttranslational modifications, and to determine the presence of possible splice variants. For the comparative analysis of chemically or post-translationally modified proteins, two samples are digested proteolytically and the resulting peptides from each are then converted to d0- and d3-methyl esters, respectively. The two samples are then mixed together and analyzed by nano flow HPLC interfaced to electrospray ionization on tandem linear ion trap-Fourier transform (LTQ-FTMS) or -orbitrap mass spectrometers. These instruments operate with a resolution in excess of 10,000, measure masses to three decimal places, and record the molecular masses of peptides in each sample at the high attomole level. For the analysis of phosphorylated proteins, immobilized metal affinity chromatography (IMAC) is employed to enrich the sample for phosphopeptides prior to analysis by nanoflow HPLC.3 The above approaches will be illustrated with examples from research involving comparative analysis of: (a) phosphorylation on proteins involved in cell migration,3 (b) post-translational modifications on histones that control gene expression, gene silencing, DNA damage repair, recombination, etc.,4 and (c) phosphopeptides presented to the immune system on cancer cells that might be used as potential cancer vaccines or therapeutics.5

1. J. E. Syka, J. J. Coon, M. J. Schroeder, J. Shabanowitz and D. F. Hunt, Proc. Natl. Acad. Sci. U. S. A. 101, 9528 (2004).
2. J. J. Coon, B. Ueberheide, J. E. Syka, D. D. Dryhurst, J. Ausio, J. Shabanowitz and D. F. Hunt, Proc. Natl. Acad. Sci. U. S. A. 102, 9463 (2005).
3. M. J. Schroeder, D. J. Webb, J. Shabanowitz, A. F. Horwitz and D. F. Hunt, J. Proteome Res. 4, 1832 (2005).
4. W. Fischle, B. S. Tseng, H. L. Dormann, B. M. Ueberheide, B. A. Garcia, J. Shabanowitz, D. F. Hunt, H. Funabiki and C. D. Allis, Nature 438, 1116 (2005).
5. Identification of Class I MHC Associated Phosphopeptides as Targets for Cancer Immunotherapy, Science submitted.