Frauke Textor (Freiburg / DE), Larissa Meyer (Freiburg / DE), Bettina Wehrle (Freiburg / DE), Stefan Tholen (Freiburg / DE), Matthias Fahrner (Freiburg / DE), Cornelius Miething (Freiburg / DE), Oliver Schilling (Freiburg / DE)
Background
Multiple gene fusions have been associated with tumor development and prognosis and in some cases are characteristic for certain malignancies. These gene fusions can result in hybrid proteins that may contain functional domains of the individual fusion partners. Molecular diagnostics of gene fusion events mainly relies on nucleic acid-based sequencing approaches and can be complemented by immunohistochemical (IHC) staining. However, IHC is antibody-dependent and serves only as a surrogate approach by detecting increased levels of either fusion proteins or upregulation of downstream signaling. Additionally, many anticancer drugs target proteins, so a deep insight into these hybrid proteins would enhance patient-specific molecular diagnostics. Therefore, we set out to establish an antibody-independent LC-MS/MS-based approach for directly detecting the fusion proteins.
Methods
In a proof-of-concept experiment, three cell lines with stable gene fusion mutations were cultured and the hybrid proteins were detected by western blot. The fusion proteins were purified and digested using the S-Trap protocol. The resulting peptides were analyzed on a TIMS-TOF flex mass spectrometer using data-independent acquisition (DIA) in parallel accumulation-serial fragmentation (PASEF) mode. The results were analyzed using DIA-NN and compared to a human spectral library that included amino acid sequences of the hybrid proteins.
Results and Outlook
We detect hybrid proteins in all three cell lines using western blot as well as our LC-MS/MS-based approach. Furthermore, in our LC-MS/MS approach we detect peptides that cover the actual gene fusion site. Hence, our approach provides an antibody-independent alternative for the direct assessment of gene fusion events and paves the way for more detailed molecular characterization of gene fusion-driven malignancies. Future experiments aim to develop a spectral library based on previously described and most common gene fusions. This library will be utilized to investigate hybrid proteins in formalin-fixed paraffin-embedded (FFPE) samples from patients with known gene fusion mutations. Establishing a robust workflow for detecting hybrid proteins in cancer patients could enhance the molecular diagnostics of gene fusion-driven malignancies, thereby improving personalized cancer treatment.