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Molecular & Cellular Proteomics 6:S34-S36, 2007.
© 2007 by The American Society for Biochemistry and Molecular Biology, Inc.

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Abstracts

Session 3

Innovative Technology for the Study of Cell Signaling

D. F. Hunt

Departments of Chemistry and Pathology, University of Virginia, Charlottesville, VA

This lecture will focus on the use of electron transfer dissociation (ETD) to identify proteins and to characterize their post-translational modifications on a chromatographic time scale. In this experiment, proteins are fractionated by nano-flow HPLC, converted to gas-phase, positive ions by electrospray ionization, and allowed to react with fluoranthene radical anions inside a linear trap mass spectrometer. Electron transfer to the multiply charged protein promotes random fragmentation of amide bonds along the protein backbone. Multiply charged fragment ions are then de-protonated in a second ion/ion reaction with the carboxylate anion of benzoic acid. MS and ETD-MS/MS spectra are recorded every 500 ms. The m/z values for the resulting singly, doubly, and triply charged ions are used to read a sequence of 15–60 amino acids at both the N and C termini of the protein. This information, along with 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 post-translational modifications, and to determine the presence of possible splice variants.

Presented first will be results of studies to characterize post-translational modifications that involve acetylation, methylation and O-GlcNAcylation.

Part two of the presentation will describe results of studies to define sites of phosphorylation that regulate the formation of focal adhesions involved in cell migration. For this work we employ immobilized metal affinity chromatography (IMAC) to identify phosphorylation sites present at levels as low as 0.1% of the parent protein. Stable isotopes are employed to follow changes in site usage as a function of cellular perturbation. Further information on this topic is available at cellmigration.org (site guide, phosphoproteomics).

Part three of the presentation will focus on signaling between cancer cells and cytotoxic killer cells by the class I, antigen-processing pathway. Since signal transduction pathways in cancer cells are highly dysregulated, we hypothesized that this might manifest itself in the presentation of unique phosphopeptides by the cancers to the immune system in association with Class I MHC molecules. By using a combination of IMAC, stable isotope labeling, and nano-flow HPLC-tandem mass spectrometry, we are able to detect cancer-specific, Class I phosphopeptides present at levels as low as 1–5 copies per cell. Recent studies show that the Class II antigen processing pathway also presents phosphopeptides. Results of studies on melanoma, ovarian, breast, and lymphoma cancers will be described.

References

1. Syka, J. E. P., Coon, J. J., Schroeder, M. J., Shabanowitz, J., and Hunt, D.F. (2004) Proc Natl Acad Sci USA 101, 9528–9533.

2. Coon, J. J., Ueberheide B., Syka, J. E. P. Dryhurst, D. D., Ausio, J., Shabanowitz, J., and Hunt, D. F. (2005) Proc Natl Acad Sci USA 102, 9463–9468.

3. Syka, J. E. P., Marto, J. A., Bai, D. L., Hornung, S. Senki, M.W., Schwartz, J. C, Ueberheide B., Garcia, B. A., Busby, S. A., Muratore, T., Shabanowitz, J., and Hunt, D.F. (2004) J Proteome Res 3, 621–626.

4. Fischle, W., Tseng, B. S. Dormann, H., Ueberheide B., Garcia, B. A., Shabanowitz, J., Hunt, D.F., Funabiki, H., and Allis, C. D. (2005) Nature 438, 1116–1122.

5. Zarling, A. L., Polefrone, J. M., Evans, A. M., Mikesh, L. M., Shabanowitz, J., Lewis, A. T., Engelhard, V. H., and Hunt, D.F. (2006) Proc Natl Acad Sci USA 103, 14889–14894.

6. Chi, A., Bai, D. L., Geer, L. Y., Shabanowitz, J., and Hunt, D. F. (2007) Int. J. Mass Spectrom. 2007, 259, 197–203.

This research was supported by NIH grants, GM-37537, AI-33993, and U54 GM-64346.

3.2

TiMAC—A New Phosphoproteomic Strategy for the Separation of Mono- from Multiply Phosphorylated Peptides Combined with Optimized Tandem MS Mass Spectrometric Analysis

T. E. Thingholm and M. R. Larsen

Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark

The ability to selectively isolate and analyze the complete complement of phosphorylated peptides from complex peptide mixture is essential for performing comprehensive phosphoproteomics. However, despite the numerous strategies used in modern phosphoproteomics including the use of IMAC, TiO₂ chromatography or phosphoramidate chemistry for affinity enrichment of phosphorylated peptides prior to mass spectrometric analysis no single method is able to provide a complete coverage of the phosphoproteome of a given sample. This has recently been shown by [Bodenmiller et al., 2007) Nat. Methods]. One of the reasons for this large discrimination between the strategies is most likely that phosphopeptides are very different both with respect to amino acid contents and number of phosphate groups attached to each peptide, providing a different binding affinity to the chelating material. In addition, the micro-environment in which a phosphate group is located, represented by amino acid functional groups, will eventually have a large influence on the physio-chemical properties of the resulting phosphopeptide and thereby also contribute to the difference in the binding affinity to different chelating material. The comprehensive analysis of phosphopeptides is furthermore compromised by common mass spectrometric instrumentation which favors the analysis of mono-phosphorylated peptides over multiply phosphorylated peptides due to ion suppression effects and inadequate peptide fragmentation.

To circumvent some of the problems in large scale phosphoproteomics of low amount of sample, listed above, we have developed a new method, TiMAC, which combine the advantages of IMAC and TiO₂ chromatography into one strategy without the use of additional material. We show that by using the TiMAC strategy we are able to separate mono-phosphorylated peptides from the multiply phosphorylated peptides prior to MS analysis. The separation of the two species allowed us to optimize the subsequent pdMS³ experiment to favor analysis of either mono-phosphorylated or multiply phosphorylated peptides. When applying this new method to a total of 120 µg protein from human mesenchymal stem cells more than twice the number of phosphorylation sites was identified compared to an optimized TiO₂ protocol.

3.3

Proteomics Study of a Steroid Signal Transduction Pathway in Plants

W. Tang¹, Z. Deng¹, J. A. Oses-Prieto², X. Zhang², N. Suzuki², S. Guan², R. J. Chalkley², Y. Sun³, A. L. Burlingame², and Z.-Y. Wang¹

¹Department of Plant Biology, Carnegie Institution, Stanford, CA; ²Mass Spectrometry Facility, Department of Pharmaceutical Chemistry, University of California, San Francisco CA; ³Institute of Molecular Cell Biology, Hebei Normal University, Shijiazhuang, Hebei, China

Brassinosteroid (BR) is a steroid hormone that plays essential roles in plant growth and development. BR binds to the extracellular domain of the receptor-like kinase BRI1 to activate its kinase activity and downstream signal transduction, which involves another receptor-like kinase (BAK1), a GSK3-like kinase (BIN2), and nuclear transcription factors (BZR1 and BZR2/BES1). BR-binding induces BRI1-BAK1 dimerization and receptor kinase activation, leading to inhibition of BIN2 and dephosphorylation of the BZR1 and BZR2/BES1 transcription factors. When BR levels are low, BIN2 phosphorylates BZR1 and BZR2/BES1 to inhibit their nuclear accumulation and DNA binding. Cytoplasmic retention of phosphorylated BZR1 and BZR2 is mediated by the phosphopeptide-binding 14-3-3 proteins. How the receptor kinases regulate BIN2 activity remains the only major gap in our understanding of the pathway. In order to identify additional proteins involved in BR signal transduction and BR-regulated physiological response, we used two-dimensional difference gel electrophoresis (2-D DIGE) and LC-MS/MS to identify BR-regulated proteins in total protein, plasma membrane (PM), and phosphoprotein fractions. We demonstrate that prefractionation is critical for identifying signal transduction components using 2-D DIGE. We also used affinity purification and MS to identify BZR1-interacting proteins. These studies identified both proteins that mediate downstream growth responses and early BR-response proteins that mediate BR signal transduction. Further studies have demonstrated the signaling function and direct interaction with known components of the BR pathway. Our proteomic studies have filled the gaps in the genetically defined BR signal transduction pathway, establishing the BR pathway as one of the best-understood signal transduction pathways from cell surface receptors to nuclear gene expression for plants as well as for steroid hormones.

3.4

Comprehensive Phosphoproteome Analyses of Leukemic Hematopoietic Stem Cells to Uncover the Molecular Basis of Self-Renewal

M. Trost¹, O. Hérault¹, M. Marcantonio^1,2, A. Faubert¹, C. Pomies¹, G. Sauvageau¹, and P. Thibault^1,2,3

¹Institute of Research in Immunology and Cancer, ²Department of Biochemistry, ³Department of Chemistry, Université de Montréal, Montréal, QC, Canada

Hematopoietic stem cells (HSC) maintain blood formation throughout life time and are typified by their ability to self-renew indefinitely and differentiate into myeloid and lymphoid blood cell lineages. Pathways regulating the maintenance of adult HSC remain poorly understood primarily due to the inability of expanding HSC in vitro and by the difficulty of isolating pure in vivo stem cells in sufficient amount. Significant advances in HSC research have been obtained through the development of different leukemia model systems, and our group previously reported the isolation of HSC with self-renewal capacity by introducing oncogenes into highly purified cell populations (Ska+, C-Kit+, Lyn–) (1,2). In this study, we compared the phosphoproteome of mice leukemia model systems showing different frequency of self renewal to investigate specific cell signaling events associated to their distinctive phenotypes. Nuclear and cytosol extracts from 10⁸ cells of leukemic HSC and normal granulocytes from mouse models were were lysed and centrifuged to isolate nuclear and cytosol protein extracts. Phosphopeptides were enriched using TiO₂ affinity media and analyzed by 1D and 2D LC-MS/MS on an Orbitrap mass spectrometer. In-house software was used to obtain expression profiles and MS/MS spectra were searched against the IPI database using Mascot. On-line 2D-LC-MS/MS provided enhanced sample loading and capacity for phosphopeptide identification with 2915 different phosphopeptides assigned to 1117 unique proteins. Comparison of phosphopeptide abundances in nuclear and cytosol extracts of HSC enabled the identification of phosphoproteins showing cellular translocation including kinases (e.g. Dyrk 1A, STK 10) and low abundance transcription factors (e.g. ETV6, ATRX) that were also correlated with leukemic stem cells of different self-renewal capacity. This study will highlight the analytical advantages of the present approach to identify cellular markers of self renewal in HSC mouse model systems of leukemia.

References

1. A. Mamo, et al. (2006) Blood 108, 622–629.

2. J. K. Krosl, A. Faubert, and G. Sauvageau (2004) Hematol. J. 5, S118–S121

3.5

New Technology to Detect and Monitor the Post-translational Modification Events that Commit Human Embryonic Stem Cells to Exit the Pluripotent State

D. L. Swaney¹, G. C. McAlister¹, D. Phanstiel¹, J. Brumbaugh², J. Keith¹, S. B. Ficarro⁴, X. Feng³, V. Ruotti⁵, R. Stewart⁵, J. A. Thomson³, T. Berggren⁵, and J. J. Coon^1,2

Departments of ¹Chemistry, ²Biomolecular Chemistry, and ³Anatomy, University of Wisconsin, Madison, WI; ⁴Department of Genomics, Novartis Foundation, San Diego, CA; ⁵WiCell Research Institute, Madison, WI

We describe the use of new mass spectrometry technology—electron transfer dissociation coupled with ultra high resolution mass analysis (orbitrap)—to discover and quantify the post-translational modification events that signal human ES cells to exit the pluripotent state. Human ES cells are grown in feeder-independent, chemically defined (TeSR1) culture and exposed to doses of various differentiating agents over multiple timepoints. Following treatment, the cells are lysed, the proteins isolated, and enzymatically digested. Using affinity chromatography (IMAC) coupled with ETD-MS/MS on our ETD-enabled orbitrap we have discovered nearly 4000 sites of phosphorylation in human ES cells. Next, we have characterized the modification status of histone H4 in pluripotent and differentiating human ES cells. This work reveals no less than 70 unique histone H4 modification patterns exist in ES cells and that these patterns change upon differentiation. Finally, we describe new technological capabilities afforded by the implementation of ETD on the hybrid orbitrap mass spectrometer. For example, we have designed a decision tree-based data-dependent method that automatically selects whether to perform CAD or ETD, based on the precursor charge, mass and abundance, and whether to perform mass analysis at high or low resolving power. For complex mixture analysis this method nearly doubles the probability that any given MS/MS event will lead to a successful identification.

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