An enormous amount of research effort has been devoted to biomarker discovery and validation. With the completion of the human genome, proteomics is now playing an increasing role in this search for new and better biomarkers. Here, what leads to successful biomarker development is reviewed and how these features may be applied in the context of proteomic biomarker research is considered. The "fit-for-purpose" approach to biomarker development suggests that untargeted proteomic approaches may be better suited for early stages of biomarker discovery, while targeted approaches are preferred for validation and implementation. A systematic screening of published biomarker articles using MS-based proteomics reveals that while both targeted and untargeted technologies are used in proteomic biomarker development, most researchers do not combine these approaches. i) The reasons for this discrepancy, (ii) how proteomic technologies can overcome technical challenges that seem to limit their translation into the clinic, and (iii) how MS can improve, complement, or replace existing clinically important assays in the future are discussed.

Hotspot testing for activating KRAS mutations is used in precision oncology to select colorectal cancer (CRC) patients who are eligible for anti-EGFR treatment. However, even for KRAS wildtype tumors anti-EGFR response rates are <30%, while mutated-KRAS does not entirely rule out response, indicating the need for improved patient stratification. We performed proteogenomic phenotyping of KRAS wildtype and KRAS G12V CRC liver metastases (mCRC). Among >9000 proteins we detected considerable expression changes including numerous proteins involved in progression and resistance in CRC. We identified peptides representing a number of predicted somatic mutations, including KRAS G12V . For eight of these, we developed a multiplexed parallel reaction monitoring (PRM) mass spectrometry assay to precisely quantify the mutated and canonical protein variants. This allowed phenotyping of eight mCRC tumors and six paired healthy tissues, by determining mutation rates on the protein level. Total KRAS expression varied between tumors (0.47-1.01 fmol/µg total protein) and healthy tissues (0.13-0.64 fmol/µg). In KRAS G12V -mCRC, G12V-mutation levels were 42-100%, while one patient had only 10% KRAS G12V but 90% KRAS wildtype . This might represent a missed therapeutic opportunity: based on hotspot sequencing, the patient was excluded from anti-EGFR treatment and instead received chemotherapy, while PRM-based tumor-phenotyping indicates the patient might have benefitted from anti-EGFR therapy.

The tumor suppressor PTEN is the main negative regulator of PI3K/AKT/mTOR signaling and is commonly found downregulated in breast cancer (BC). Conflicting data from conventional immunoassays such as immunohistochemistry (IHC) has sparked controversy about PTEN’s role as a prognostic and predictive biomarker in BC, which can be largely attributed to the lack of specificity, sensitivity, and interlaboratory standardization. Here, we present a fully standardized, highly sensitive, robust microflow immuno-MRM (iMRM) assay that enables precise quantitation of PTEN concentrations in cells and fresh frozen (FF) and formalin-fixed paraffin-embedded (FFPE) tissues, down to 0.1 fmol/10 μg of extracted protein, with high interday and intraday precision (CV 6.3%). PTEN protein levels in BC PDX samples that were determined by iMRM correlate well with semiquantitative IHC and WB data. iMRM, however, allowed the precise quantitation of PTENeven in samples that were deemed to be PTEN negative by IHC or western blot (WB)while requiring substantially less tumor tissue than WB. This is particularly relevant because the extent of PTEN downregulation in tumors has been shown to correlate with severity. Our standardized and robust workflow includes an 11 min microflow LC-MRM analysis on a triple-quadrupole MS and thus provides a much needed tool for the study of PTEN as a potential biomarker for BC.