1. Monitoring proteins relevant to tumor angiogenesis and tumor progression by proteomic analysis of biofluids and tissue from cancer patients
In collaboration with the groups of C. Ruegg (CePO, LCC, UNIL) M. Hegi (Lab. of Tumor Biology and Genetics, CHUV), R. Stupp (CePO, LCC, CHUV, UNIL), B. Domon and R. Aebersold (IMSB- ETH)
We are following two approaches:
i) Targeted analysis of molecules in biofluids and tissues. In a hypothesis-driven approach we will accurately identify and quantify selected proteins in CSF and serum from patients with multiple glioblastoma or other solid tumors (e.g. colon, breast, head and neck cancers). We will detect molecules that have been previously identified by a single biomarker approach (e.g. VEGF-A, MMP-9) and molecules that have been identified as potential candidates of angiogenesis or tumor progression in gene expression studies (e.g. Cyr61/CCN1; GDF3, GDF-15, EPSTI-1). We will employ a quantitative proteomics approach developed by Aebersold which is based on the selection and chemical synthesis of isotopically labeled reference peptides corresponding to peptides generated from the native protein by tryptic digestion. Since the proteins of interest are glycoproteins, we will use the glycoprotein enrichment method which is developed by the same group and further analysis by LC-ESI MS in SRM/MRM mode in the presence of the reference peptide. This approach has been proven suitable for detecting a panel of candidate biomarkers in clinical samples.
ii) Global protein profiling in biofluids and cancer tissues. Once this first approach has been successfully established, we plan to analyze tumor tissue and biofluids for global protein expression profiles (signatures) specific to the tumor, its propensity to progress (patient survival) and (possibly) its response to therapy. We will study only patients for which clinical follow up and/or patient outcome is known. The proposed strategy involves fractionation of the material to ensure characterization of the different types of components (e.g. small vs large proteins). After immunodepletion of the high abundant proteins, samples will be reduced, alkylated and digested by trypsin and generated peptides separated by 2-D LC and analyzed by MALDI-TOF/TOF. Quantitative differences in the proteins present in tumor tissue or serum/CSF samples will be initially determined through comparison of the peak intensities of their constituent peptides/proteins. Later, when specific peaks have been characterized as interesting, we will quantitate the corresponding proteins by the use of isotopically labeled reference peptides (see above).
2. Mass Spectrometry-based strategy to measure alteration of glycosylation
In collaboration with Bruno Domon, ETH - IMSB.
The aim of this project is to detect the alteration of the glycosylation pattern in the serum of cancer patients by differential analyses using stable isotope labelling. The method developed includes cleavage of N-linked glycans from glycoproteins in serum using PNGase F, and derivatization of the glycans with 2-aminopyridine (or an isotopically labeled variant). The reductively aminated samples are mixed, chemically desialylated and analyzed on a quadrupole time-of-flight system coupled to a micro-HPLC system and a microfluidic device. The different glycoforms are assigned based on their molecular mass, and their structure is confirmed by their MS/MS patterns. The relative ratio of the signal of one glycoform reflects the abundance of the glycan in the two samples.
We will apply this method to the relative quantification of glycans in the serum of patients with colon, breast cancer and controls. In addition to the analysis of the glycans from the whole serum, glycan patterns will also be analyzed after the depletion of the major high abundant proteins in serum. This will provide information about N-glycans from low abundant proteins which may include membrane surface proteins that play an important role in cancer development.
3. The identification of new peptide tumor antigens derived from alternatively spliced variants expressed specifically in neoplastic cells (NCCR)
Within this project, our main focus is the analysis (separation and identification) and quantitation of potential HLA class I-restricted ligands generated by proteasomal degradation of precursor peptides encoded by alternatively expressed exons. The proteolytic activities of other peptidases involved in the antigen processing pathway will also be studied and analyzed.