Steen Laboratory

Research

The development of novel and improved mass spectrometric and proteomic methods for the analysis of complex protein mixtures and the detailed characterization of proteins within these mixtures, including the analysis of post-translational protein modifications (PTMs) and the quantitation of protein abundances and their modifications.
Mass Spectrometry and proteomic methods are rapidly evolving such that new instrumentation and new analytical methods developed in the wet laborotory can give rise to more efficient identification of modifications or the identification of new modifications. There are over 200 different post-translational and co-translational protein modifications. Many of these modifications are rare or not reported often as there are difficult to detect or are found under very specific physiological conditions. However, some PTMs are ubiquitous. These include protein phosphorylation, glycosylation, acylation and processing such as proteolytic cleavage. We are interested in developing novel methods of enrichment and detection using masspectrometric methods. Determination of PTM's represents a major challenge because only a limited portion of the molecules of a given protein in the cell might be modified and the modifications will often be located on different positions of the molecule depending on its actual functional state. This results in a heterogeneous population of the given protein this is typical for many proteins. The modifying group may also be highly heterogeneous as is typically seen for glycosylation where a given site may be occupied with more than twenty different glycan structures. As a consequence, highly sensitive and selective analytical methodology is required for complete analysis of all the post-translationally modified forms of the proteins present in a cell or a tissue. We are currently developing on both analytical methods for specific modification such as glycosylation, g-carboxylation and phosphorylation.

Development of instrument platforms for the detection and identification of disease-markers with special emphasis on pediatric diseases.
Proteomic biomarkers can be specific physical traits that can be both qualitative and quantitative. As such these measurements can indicate the effects or progress of a disease or condition. These biomarkers can be very useful for diagnosis i.e. deducing the grade of a tumor, treatment and evaluating the effectiveness of a treatment. Instead looking for biomarkers in highly complex protein mixtures from cancer cells, focusing on the cell cycle protein machinery for biomarkers appears to be more promising from our preliminary studies.

The use of mass spectrometry to identify mechanisms of cell cycle control.
The APC is E3 ubiquitin ligase that is essential to cell division and differentiation. It plays a central role in the metaphase to anaphase transition. The APC is inhibited by the spindle checkpoint complex unless all chromosomes are attached to the mitotic spindles thus ensuring the accurate division of genetic material. The APC will be isolated from different cancer cell lines before and after treatments with various drugs either individually or in combination. These isolated complexes will then be subjected to detailed analysis using mass spectrometry. Mass spectrometry is powerful technique in that the detailed analysis of protein sequences and the unambiguous localization of protein modifications can be obtained. As these modifications are utilized by cells to fine tune the functions of the different proteins, it is of pivotal importance to unambiguously map and analyze these modifications before they can be studied in detail. This is especially the case for proteins and protein complexes such as the APC that are involved in the regulation of cell proliferation and differentiation, two processes intimately associated with tumor genesis.

Development of methods for the stable isotope-free mass spectrometric quantitiation of protein phosphorylation.
One of the known - or perhaps the - main regulatory components in protein function is reversible protein phosphorylation. Although the localization of phosphorylation sites is in itself challenging enough, it becomes increasingly apparent that the extent of phosphorylation is of crucial importance, and hence every phosphorylation study should also show quantitative information. Apart from 32P-labeling and quantitative Western blotting with phospho-specific antibodies, mass spectrometry is currently the only universal method which allows the localization of phosphorylation sites and the acquisition of 'residue-resolved' quantitative information. Quantitative mass spectrometry normally utilizes stable isotope labeling of some kind, but my recent studies showed that mass spectrometry is sufficiently quantitative if robust normalization procedures are applied to account for run-to-run variations and varying amounts of starting material. Thus mass spectrometry is able to provide the desired quantitative information without stable isotope labels. This allows the relative quantitation of protein phosphorylation, i.e. quantitation of the changes in phosphorylation stoichiometry, as well as the absolute quantitation, i.e. determination of the degree of phosphorylation, in a much faster, cheaper and more generally applicable way. Furthermore this approach is not limited to protein phosphorylation but protein modifications in general.