Summary
Comprehensive proteome analysis of cellular processes on a large scale is limited in some way for many of the currently available methods like MUDPIT or two-dimensional electrophoresis. We want to exploit an efficient and high-throughput proteomics method, unbiased for separation of extreme protein characteristics and at the same time allowing separation of protein isoforms. The approach will be based on protein separation by chromatography in multiple dimensions (MDLC). Consecutive separation dimensions will be explored, like SAX, SCX, reversed phase chromatography. In combination with quantification methods such as fluorescent- and stable isotope labeling, this will enable us to establish differential protein levels from 2 or more samples.
This approach will be applied to unravel the human erythrocyte proteome. Recently, we succeeded in developing a new method that efficiently removes high abundant proteins, hemoglobin and carbonic anhydrase, which improved the number of identified proteins over 4 times. Other, more generic depletion methods using peptide libraries coupled to beads will be explored for removal of high abundant erythrocyte membrane proteins. By this combined approach of efficient depletion for abundant proteins and successive fractionation of complex proteomes, the dynamic range of the proteome can be dramatically increased.
We aim at unravelling the protein molecular background of yet unknown erythrocyte disorders causing anemia. This work is performed in collaboration with the Laboratory Clinical Chemistry and Haematology (University Medical Center Utrecht, Utrecht). For approximately 50% of the patients, the molecular defects in erythrocyte membranes and –metabolism are not known, which hampers proper diagnosis, treatment and counselling of patients. The department of Clinical Chemistry and Haematology at UMCU has a long standing track record in hereditary red cell research and is an international reference site for red cell diagnostics. This proposed research methodology has great value, since it opens up a totally new and very promising area of diagnostics and research in erythrocytes. In addition, eventually new diagnostic tests can be developed in collaboration with  companies, making diagnostics of these disorders internationally more feasible.