Abstract 4577: Comparative proteomic biomarker screening in laser microdissected tissues from colon adenoma and cancer

Colorectal (CRC) malignancies rank world wide at the third place for tumor diseases and account for an annual mortality rate of 492 000 cases. Although several molecular events are known to be involved in the transition from normal tissue to adenoma and finally to undifferentiated carcinoma it remains a challenge to discover new and more reliable biomarkers for diagnosis, prognosis and prediction of outcome. Towards this end a study was designed to identify potential biomarkers which are associated with the molecular events leading from epithelial adenoma to the early stages of carcinoma. A new biomarker discovery strategy was developed to combine the cell specificity and selectivity of laser capture microdissection (LCM) with the resolution power and sensitivity of liquid-chromatography (LC)-matrix-assisted-laser-desorption/ionization mass spectrometry (LC-MALDI-MS). We carefully selected a group of closely matched patients (n = 10 for each group) afflicted with epithelial adenoma (high dysplasia) or early stages of carcinoma (stage I) and used the derived normal as well as the matched tumor tissue samples to reveal protein expression differences. According to this LC-MALDI-MS strategy microdissected cells (around 10 000 cells) were lysed and extracted proteins were digested with Trypsin. Obtained peptides were separated by capillary reversed phase HPLC (Agilent). The resulting LC-fractions (300) were spotted on prespotted AnchorChip targets (PAC, Bruker) and tryptic fragments subsequently detected by reflector MALDI-MS (ultraflex III, Bruker) measurements. This generated between 5000-7000 ion signals ranging from m/z 800 to 4000. Differential peptide analysis was then performed with the goal to discover robust and significant expression differences between patient groups. Therefore, only m/z ions displaying a minimum twofold difference and a p-value of 0.01 between the tissue samples were considered for further analysis. The selected peptides were subsequently fragmented by MS/MS experiments to reveal their primary sequence and protein identity. Our targeted biomarker discovery approach resulted in the identification of more than 30 biomarker candidates which act in diverse cellular functions and can now be linked to early events (e.g. adenoma vs. normal tissue) or later events (e.g. carcinoma vs. normal) of tumor progression. Currently these biomarkers are validated using antibody based assays to further analyze their potential as markers in a clinical