Contribution of the proteomics platform to molecular imaging targets?

Our role in the “KeyMarker” project is to characterize specific biomarkers involved into different pathologies. These pathologies are linked to the concerned associates of the project. We proposed to use original proteomic methods to identify these biomarkers. The first part of our work is to characterize these predictive markers into the plasma of women treated for breast cancer chemotherapy. These potential biomarkers are present into the plasma as low abundant proteins. They are entirely masked by highly abundant proteins and significantly complicate the discovery process by limiting the detection of low abundance proteins.

We proposed to use protein depletion to remove highly abundant proteins from plasma with Ig-based immunoaffinity columns. These columns capture the more abundant proteins from human biofluids such as plasma. We tested 2 different immunoaffinity colums. One is designed to remove twelve high-abundant proteins and the other can remove twenty high-abundant proteins. The depleted fraction is analyzed by two independent strategies. The first one is two-dimensional gel electrophoresis (2D gel) followed by mass spectrometry for protein identification (MALDI/MS). The second one is one-dimensional gel electrophoresis (SDS-PAGE gel) followed by liquid chromatography and mass spectrometry (1D-LC/ESI). Removal of high-abundant proteins enables improved resolution for one-dimensional and two-dimensional gel electrophoresis. These complementary strategies should provide us a maximum number of low abundant proteins.

Type I and type II diabetes are pathologies characterized by abnormal apoptosis of beta-cells in pancreatic islets. Apoptosis is a physiological process triggering programmed cell dead. -cell apoptosis can be induced by the inflammatory mediator cytokine interleukin (IL)-1 β. This (IL)-1 β is used in vitro to promote -cell « decision » to undergo apoptosis. Cell lines or primary cell cultures of rat pancreatic beta-cells are cell models employ by Pr. Decio Eizirik. This group identified ~700 genes and EST that are modulated in extensive microarray experiments.
Following these studies, Pr. Decio Eizirik wishes to use the experience from the Pr. Edwin De Pauw’s laboratory to characterize protein expression pattern of -cell undergoing apoptosis or not. To be able to use these specific -cell protein biomarkers for medical imaging (PET scan: Positron Emission Tomography), these makers should be accessible to antibodies used for imaging and could be potential therapeutic targets. Pr. Edwin De Pauw suggest to make a sub-proteome containing beta-cell transmembrane proteins treated or not with interleukin (IL)-1 β. Protein fractionation will be performed by labelling of their extracellular domains with biotin. Biotinylated proteins can react with streptavidin link to a streptavidin-sepharose column. Trypsin could be use to release labelled proteins. Biotinylated peptides will be identified after separation by 2D-LC and mass spectrometry (ESI).

Type I and type II diabetes are pathologies characterized by abnormal apoptosis of b-cells in pancreatic islets. Apoptosis is a physiological process triggering programmed cell dead. b-cell apoptosis can be induced by the inflammatory mediator cytokine interleukin (IL)-1 β. (IL)-1 β is used in vitro to promote cell cultures of rat pancreatic b-cells « decision » to undergo apoptosis. Pr. D. Eizirik identified ~700 genes and EST that are modulated by (IL)-1 β in microarray experiments. Following these studies, Pr. D. Eizirik wishes to use the experience from the Pr. E. De Pauw’s laboratory to characterize protein expression pattern of b-cell undergoing apoptosis or not. To be able to use these specific b-cell protein biomarkers for medical imaging, they should be accessible to antibodies. Pr. E. De Pauw suggest to make a sub-proteome containing b-cell transmembrane proteins treated or not with interleukin (IL)-1 β. Protein fractionation will be performed by labelling of their extracellular domains with biotin. Biotinylated proteins can react with streptavidin link to a streptavidin-sepharose resin. Trypsin could be use to release labelled proteins. Biotinylated peptides will be identified after separation by 2D-LC and mass spectrometry (ESI).

Outlook

Our goal is to use molecular biomarker for medical imaging (PET scan: Positron Emission Tomography). Diagnostic imaging procedures are crucial for further treatment planning in many diseases.
Pr. D. Eizirik wishes to characterize specific biomarkers induced during apoptosis process into the  cells. These cells are undergoing apoptosis during type I and II diabetes. To use these markers for medical imaging, the marker should be a plasma cell membrane protein and should be accessible to the antibody. Pr. D. Eizirik will use the proteomic plateform at the Giga center to identify potential molecular imaging targets.