Background and AimProteome profiling still has to enter the realm of clinical pathology on a wider scale. To facilitate the integration of mass spectrometry-based proteomics into routinely executed workflows of molecular diagnostics, the usage of formalin-fixated, paraffin-embedded (FFPE) specimens is key, as this is the mainstay approach to sample handling and conservation in clinical pathology. In molecular diagnostics, the quest for multiplexed protein analytics and deep proteome-level insight into therapy-guiding markers is gaining momentum and is ideally based on the tight integration into pre-existing diagnostic workflows. These approaches extend beyond oncology to include inflammatory diseases and amyloidosis typing. Methods

We have established the routine in-depth proteomic investigation using FFPE tissue samples as well as other clinically relevant sample specimens from as little as 0,01 mm³ of tissue rendering most patient-derived samples accesible, including small needle biopsies. Additional laser capture microdissection (LMD) enables tissue and cell-specific proteome profiling in a spatial context. Sample preparation and LC-MS/MS data analysis have been standardized and automated while robust LC-MS/MS instrumentation and acquisition strategies enable fast and reproducible data acquisition to meet the needs for clinical and diagnostic applications. High-coverage phospho-proteomic profiling from FFPE specimens has been established.

Results

We have integrated mass-spectrometry-based proteomics and phosphoproteomics into the diagnostic portfolio of the Molecular Tumor Board (MTB) Freiburg. To date, we have provided proteome profiling for > 250 MTB cases to support clinical decision-making. The usage of single-shot, label-free quantitation approaches has provided the required flexibility and speed. In addition, we have assembled an in-house reference dataset of > 3000 proteomes covering a variety of malignancies and leading towards a growing reference database of cancer-related proteomes as well as inflammatory diseases. Comparing individual cases against molecular- and/or entity-matched references might facilitate the identification of patient-specific tumor-driving factors. The proteomic detection of clinically relevant sequence variants such as KRAS G12C or BRAF V600E has proven applicable to foster proteo-genomic corroboration and target prioritization. Phosphoproteomics has corroborated oncogenic signaling activity in select MTB cases. Integration of these approaches into the Centers for Personalized Medicine Baden-Württemberg facilitated diagnostically meaningful interlaboratory tests that underline the robustness and reproducibility of proteome profiling.

Conclusion

MS-based clinical proteomics and proteogenomics has advanced as a sensitive and robust approach to complement current molecular profiling and paves the way towards precision medicine.