It is a well-appreciated fact that phosphorylation is a crucial regulatory event for mitosis and many other cellular processes. However, the procedures for identifying phosphorylation sites on proteins tend to be tedious and difficult. Recent developments in mass spectrometry (MS) have revolutionized the analysis of the post-translational modifications on individual proteins. In this Work Package, we will build on this expertise to develop phospho-MS methods with much increased sensitivity and throughput. In the course of the MitoCheck project, we will use this method to map mitosis-specific phosphorylation sites on a minimum of 20 protein complexes that are required for mitosis. Furthermore, we intend to elucidate which mitotic kinase is responsible for generating which phosphorylation site on mitotic protein complexes by employing small molecule inhibitors specific for certain kinases. Results from these studies shall significantly advance our understanding of how mitotic kinases help to orchestrate many mitotic processes by modifying different substrates.

- To develop phospho-mass spectrometry methods with increased sensitivity and throughput for identifying phosphorylation sites on mitotic proteins.

- To determine mitosis-specific phosphorylation sites on proteins required for mitosis.

- To identify which mitotic kinases are required for the generation of which phosphorylation sites on about 20 mitotic protein complexes

Jan-Michael Peters (Research Institute of Molecular Pathology, Vienna, Austria)

Richard Durbin (Wellcome Trust Sanger Institute, Hinxton, UK)

1) To develop high sensitivity and high throughput phospho-MS analysis methods

The major challenge of this task is to develop phospho-MS analysis methods with sensitivity and throughput high enough for identification of a large number of phosphorylation sites.

We previously established a standard MS protocol for phospho-analysis (Alexandru et al., Cell 105, 459-472, 2001). However, this method is not selective for phospho-peptides and data interpretation takes days or weeks for one sample, making it unsuitable for phosphorylation site identification on a large scale. We have taken an alternative MS approach whereby phosphorylated proteolytic fragments can be selectively detected on QqQ mass spectrometers, and that structure of these phosphopeptides can be determined on the same MS instrument and in a single experiment. In addition, we have optimized sample preparation, data acquisition and evaluation as well as database searching parameters. The current method (Herzog et al. Meths. Enzymol. 2005; 398:231-45) proved to be reliable, sensitive and suitable for high-throughput identification of mitosis-specific phosphorylation sites on mitotic protein complexes.

2) To map mitosis-specific phosphorylation sites on protein complexes required for mitosis.

To determine which phosphorylation sites are mitosis-specific, we will first enrich cells in interphase or mitosis by chemical treatment, isolate protein complexes of interest by immunoprecipitation or affinity purification and subject these complexes to phospho-MS analysis. The sites that are phosphorylated only in mitotic cells are deemed mitosis-specific. We have already mapped the mitosis-specific phosphorylation sites on 12 mitotic complexes. We plan to do so on a minimum of 20 protein complexes by the end of the project.

3) To identify which mitotic kinases are required for the generation of which phosphorylation sites on mitotic protein complexes

Recent discovery of several chemical inhibitors specific for particular kinases, such as Hesperadin for Aurora-B, BI 2536 for Plk1 and Roscovitine or Kenpaullone for Cdk1, has greatly enhanced our ability to manipulate the activity of kinases in living cells. For example, to determine which phosphorylation sites depend on Aurora-B activity, we will purify protein complexes from cells treated either with or without Hesperadin followed by phospho-MS analysis. A comparison of phosphorylation sites on complexes isolated either from untreated or Hesperadin-treated mitotic cells will reveal whether Aurora-B is required for the generation of these phospho-residues.

4) MS data management

As a large-scale proteomics project, MitoCheck faces numerous mass-spec data-management issues such as linking of sample preparation data to the corresponding MS data files, interactive displays allowing comparison of multiple result sets and identification of further information about the proteins of interest. We have established an integrated suite of programs that manages data during each stage of the proteomics workflow. Ongoing development includes generation of links from: (1) selected protein hit to external internet-based resources, giving sequence, function, interaction, literature and other data; (2) selected protein hit to relevant gene page in the MitoCheck database, showing information from the RNAi screen; (3) selected (phospho-) peptide hit to an MS spectrum viewer, allowing the quality of the spectrum and the peptide match to be visually evaluated.

- A systematic comparative and structural analysis of protein phosphorylation sites based on the mtcPTM database.
Jimenez JL, Hegemann B, Hutchins JR, Peters JM, Durbin R. Genome Biol. 2007 May 23; 8(5):R90 (PMID: 17521420)

- The small-molecule inhibitor BI 2536 reveals novel insights into mitotic roles of polo-like kinase 1.
Lenart P, Petronczki M, Steegmaier M, Di Fiore B, Lipp JJ, Hoffmann M, Rettig WJ, Kraut N, Peters JM.. Curr Biol. 2007 Feb 20;17(4):304-15. (PMID: 17291761)

- Application of different fragmentation techniques for the analysis of phospho-peptides using a hybrid linear ion trap-FTICR mass spectrometer.
Stingl C, Ihling C, Ammerer G, Sinz A, Mechtler K. Biochim Biophys Acta. 2006 Dec;1764(12):1842-52. (PMID: 17097932)

- Protein phosphatase 2A protects centromeric sister chromatid cohesion during meiosis I.
Riedel CG, Katis VL, Katou Y, Mori S, Itoh T, Helmhart W, Galova M, Petronczki M, Gregan J, Cetin B, Mudrak I, Ogris E, Mechtler K, Pelletier L, Buchholz F, Shirahige K, Nasmyth K (2006) Nature 441:53-61 (PMID: 16541024)

- Titanium dioxide as a chemo-affinity solid phase in offline phosphopeptide chromatography prior to HPLC-MS/MS analysis.
Mazanek M, Mitulovic G, Herzog F, Stingl C, Hutchins JRA, Peters JM and Mechtler K. Nature Protocols 2006 1:1977-87 (PMID: 17545998)

- Identification of cell cycle-dependent phosphorylation sites on the anaphase-promoting complex/cyclosome by mass spectrometry.
Herzog F, Mechtler K, Peters JM (2005). Methods Enzymol 398:231-45 (PMID: 16275332)

- Dissociation of cohesin from chromosome arms and loss of arm cohesion during early mitosis depends on phosphorylation of SA2.
Hauf S, Roitinger E, Koch B, Dittrich CM, Mechtler K, Peters JM. PLoS Biol. 2005 Mar;3(3):e69. (PMID: 15737063)