Development of alternative proteomics methods for lung cancer biomarker evaluation. (Doctoral thesis)

Lung cancer is a serious public health problem, killing millions of people around the world every year. It is the third most common cancer in Europe after colorectal cancer and breast cancer, but the deadliest since it is often detected too late. Sensitive and reliable methods for early stage detection and drug response prediction could significantly improve the therapeutic treatment and survival rate of lung cancer patients. Although tremendous efforts in biomedical research have been made over the last decades to develop novel biomarkers for cancer diagnosis and treatment response, no reliable and specific panels of biomarkers are available for early lung cancer diagnosis. During the past decade mass spectrometry (MS) based proteomics has become an important approach to biological and clinical investigations. Most MS-based protein quantification approaches generally involve trypsin digestion of the endogenous proteins in biological samples, followed by a targeted liquid chromatography-mass spectrometry (LC-MS) analysis of signature peptides indicative of the proteins of interest. Trypsin cleaves proteins C-terminal of lysine and arginine residues generating peptides that are well suited for LC-MS/MS analysis. However, despite the undeniable advantages of trypsin, the enzyme is not optimal for all types of proteomics studies due to an uneven distribution of the cleavage sites in the human proteome. During a tryptic digestion also very short non-specific peptides (< 6 amino acids) are produced resulting in a loss of sequence protein coverage. This can be problematic as the missing peptide parts could contain crucial information for disease understanding, such as specific amino acid mutations or post- translational modifications of amino acid residues. Moreover, the generation of large numbers of short peptides results in an increased background complexity which is a main limiting factor in quantification experiments as it reduces the selectivity of the measurements, which in turn affects the sensitivity of the experiments. Isotope dilution strategies are frequently employed to achieve accurate quantification of the proteins of interest using calibrated isotopically labeled peptides as internal standards. In quantitative assays, which involve the use of stable isotope-labeled standards, the reliability and the accuracy of the experiments is principally dependent on the quality of the standards. Different factors can affect the standard concentration between the synthesis and its utilization, such as incomplete solubilization, non-specific adsorption to hydrophobic surfaces or aggregation. To ensure an accurate quantification the internal standards need to be verified immediately before analysis. The purpose of this PhD thesis was to develop new proteomic approaches to remedy these limitations by using, instead of trypsin, alternative enzymes and by developing a quantification approach based on concatenated polypeptide standards containing a cleavable reporter peptide for accurate quantification.