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Already before the occurrence of pathological symptoms in the development of severe diseases changes in the proteome pattern in the plasma of patients can be observed. Therefore, to follow these pathological changes, plasma samples from several time points should be analysed before clinical symptoms arise. Future patients can be statistically expected within the pool of periodical blood donors. From the biobank (blood donor centre of the Bavarian red cross) colon cancer patients can be derived out of a pool of nearly 300,000 donors every year.
Within this project (20 individuals) plasma samples of five different time points, collected each from the same individual were depleted from 12 high abundant proteins, labelled with ICPL and separated by 2D-gelelektrophoresis. Proteins were stained with Sypro-Ruby, picked and enzymatically cleaved with Trypsin. Peptides and corresponding proteins were identified by MALDI-MS and MS/MS analysis. From several gene products up to 30 different isoforms could be found, many of them differentially regulated.
These isoforms, usually derived from one single gene, are produced due to splicing, degradation and posttranslational events. Importantly, they would not have been discriminated if GIST or if shotgun proteomics approaches would have been used. Applying these methods, the proteome would have been cleaved enzymatically before labelling. After enzymatic cleavage of similar protein species their peptides could not be reliably used for quantification since they may be obtained from differently regulated proteins. Only a few proteotypic peptides (which are specific for a single protein species) are suited for accurate quantification. However, with usual sequence coverage of far less than 50% chances are quite high to miss these peptides.
During the ICPL approach the stable isotopic label is already introduced on protein level, thereby bypassing the disadvantages of GIST approaches and maintaining the benefits of correlating protein modifications with their regulation.