Analysis of cancer related protein isoforms by mass spectrometry. (Doctoral thesis)

Cancers are frequently caused by protein modifications effecting the biochemical function resulting in abnormal expression of protein products and resulting in alterations in the cells. Consequently, the study of proteins and their variants constitute the essence of cancer proteomics aiming at the discovery of novel biomarkers. Changes at the protein level can be introduced mainly by mutations at the DNA level or by post-translational modification events. Although germ-line mutations can result in predisposition to heritable cancers, somatic mutations represent the majority of observed genomic alterations and are not found in matched normal tissues from the same patient. The ability to detect these modified biomolecules with high precision across multiple biological samples is preferentially performed by mass spectrometry based technologies due to the ability of this method to determine the molecular mass of the protein as an accurate characteristic of each protein.
Lung cancer is a heterogeneous disease that is characterized by a spectrum of somatic and genomic “driver” alterations. Profiling lung cancer includes screening for diverse prognostic and predictive biomarkers to assess the prognosis and predict the outcome of treatment. Currently in the clinical environment the treatment options for lung cancer patients are based on histology results and the stage of the tumor. Although these drug treatments demonstrated promising results, patients with an advanced disease stage have poor prognosis also due to an acquired resistance to the drugs. Therefore, EGFR deletion (delE746-A750) and point (L858R) mutations (increased or decreased sensitivity to drugs) were used as proof-of-principle for developing a targeted strategy. Furthermore, this strategy was applied on KRas as one of the Ras family isoforms (decreased sensitivity towards EGFR inhibitors) and another “driver” oncogene in NSCLC.
Quantitative mass spectrometric analyses of modified proteins in extracts from tissue samples are however challenging due to the high complexity of the samples and the requirement to detect exactly the modified part of the molecules of interest. Thus, the aim of the project is to design, implement and validate specific tests for protein variants, derived from genetic and/or post-translational changes, known to be involved in cancer formation. The limitations for quantitative mass spectrometry analyses in complex biological samples can be overcome by the use of internal standards in combination with an immuno-enrichment strategy for those proteins containing sequence variations and/or post-translational modification. Each step was carefully designed to obtain optimal datasets yielding the basis for a solid data analysis of the targeted isoforms. The comparison to the current genomic techniques underlined the importance of the investigation to be performed at the protein level. The creation of a platform with the latest technology advances in mass spectrometry can position these proteomics assays into routine applications and may find an immediate clinical function for patient stratification and ultimately for therapeutic decisions by clinicians.