Improving drug discovery with advanced targeted proteomic monitoring workflows

Proteins are key therapeutic targets due to the biochemical functions they perform within our bodies. As we continue the move to an era of precision medicine, greater understanding of proteins and protein networks and their relationships with diseases like cancer enables scientists to better identify targets for developing new therapeutics.

To fully understand these networks, analytical methods with high sensitivity, reproducibility and selectivity are needed. One of the most powerful techniques commonly used to support therapeutic research and development is mass spectrometry (MS), which is notably being applied to the characterisation of tyrosine phosphorylation (pTyr).

Current discovery and targeted MS‑based methods commonly used to monitor protein networks must compromise between broad coverage of the network of interest, reproducibility in target identification across analyses and accurate quantification. However, a novel method could remove these limitations and effectively enable target identification.

The importance of the target pTyr

pTyr plays a key role in cell signalling and is commonly dysregulated in cancer. Studying a tumour’s pTyr levels can, therefore, provide potentially useful insights for creating new therapies by identifying relevant target molecules. However, unlocking these insights is not a straightforward process.

Deep profiling of pTyr-mediated signalling requires pTyr enrichment and substantially higher sensitivity than standard phosphoproteomic or protein expression profiling approaches.

Alongside this, the ability to measure low abundance tyrosine phosphorylated peptides remains challenging for drug discovery laboratories, particularly when working with limited amounts of sample material.

The challenge with traditional targeted protein quantitation

Traditional targeted protein quantitation assays are typically based on affinity reagent-based methods, such as ELISAs, western blotting or immunohistochemistry staining. However, these techniques are limited by dynamic range, specificity, sensitivity and the small number of targets that are detectable and quantifiable. These restrictions thus affect the breadth and depth of ongoing research.

High-sensitivity MS-based pTyr methods – data‑dependent acquisition (DDA); data-independent acquisition (DIA); and parallel or multiple-reaction monitoring (PRM/MRM) – provide an attractive alternative to traditional assays, although each has its own limitations. Scientists are therefore forced to compromise between broad coverage of the pTyr network, reproducibility in target identification across analyses and accurate quantification.

For example, DDA offers deep sequencing but can result in inconsistent reproducibility of detected peptides, leading to numerous missing values across multi-analysis studies and the risk of valuable insight being overlooked. Meanwhile, PRM/MRM methods often entail a compromise between the number of peptides that can be reliably measured and sensitivity/selectivity of those measurements, restricting depth of coverage. These methods also commonly require complex method acquisition structures and peptide retention-time scheduling, which limits ease of use.

Finally, DIA methods make quantitative accuracy challenging due to both the complexity of DIA spectra and techniques demonstrating lower sensitivity than PRM approaches. The latter is a critical consideration with low abundance, tyrosine phosphorylated peptides.

A novel approach for target characterisation

To overcome the limitations of existing MS methods, a new targeted approach has been developed, as outlined in a study published in Cancer Research.¹ This pairs low input pTyr enrichment with a panel of isotopically labelled internal standard (IS) peptides to guide data acquisition of low-abundance tyrosine phosphopeptides.

This novel method addresses the challenges of DDA and other targeted MS-based approaches to provide enhanced selectivity and sensitivity. Furthermore, it increases efficiency by maximising the number of targetable peptides and simplifying assay development, since it does not rely on retention time-scheduling.

The applicability of the method was established through a study of pTyr signalling levels in human colorectal tumours, using minimal sample input.¹ Additionally, the quantitative reproducibility of this approach was realised by performing targeted pTyr profiles on three replicate in vitro samples. By using this method, scientists were able to reliably quantify several hundred commonly dysregulated pTyr targets with high accuracy, improving the robustness and usability of targeted MS assays.

The high quantitative reproducibility achieved with the method-triggered targeted workflow has many benefits, including the ability to readily analyse signalling network dynamics under various conditions. The signalling state of a patient‑derived tissue can also be compared over time as therapeutic resistance or metastases develop, which could help inform treatment options in the clinic and the development of new drug targets in the laboratory.

Furthermore, while many targeted workflows require complex method structures and customised MS platforms, all aspects of the method were performed with commercially available nano‑high performance liquid chromatography (HPLC) columns, enrichment reagents, method templates and instrumentation. Executing this workflow simply requires the IS peptide mixture and a single survey analysis to determine intensity thresholds for IS peptide triggering.

New possibilities for cancer therapies

This technique could enable rapid and reproducible pTyr profiling in both clinical and research settings, facilitating pTyr-based measurements for applications in precision medicine, including oncological therapies. In some cases, the pTyr signalling data revealed therapeutic opportunities in tumours that would have been missed by traditional biomarker analysis methods, demonstrating the power of pTyr characterisation as a complementary approach for selecting treatment strategies.

This MS method is suitable for clinical sample profiling, as it only requires 800μg of total protein as sample input material – less than a standard 14G needle biopsy. This is particularly important when sample size from a patient is limited and could prevent the necessity for more invasive biopsy procedures. Moreover, using a set of reference standards for quantitation enables comparisons across research projects and data collection sites, paving the way for large, multisite studies using pTyr levels for disease characterisation.

Extensive potential of the new approach

The targeted quantitation workflow of the new approach therefore delivers a complete MS assay from sample preparation to monitoring and quantitation of target peptides.

The method framework can also be applied to a wider panel of peptides for deeper profiling of the tyrosine phosphoproteome. Indeed, improvements to assay accuracy, reproducibility and ease of use may open new doors in research and clinical settings to support the ongoing identification of targeted therapeutics and the patients most likely to benefit from a given treatment.

Furthermore, there are wider implications for drug discovery due to its ability to support analysis of other protein networks and proteomic studies. By delivering sensitive multiplexed quantitation of many targets, the technique enables the analysis and monitoring of numerous proteins.

Ultimately, targeted profiling, either as a standalone strategy or in combination with proteogenomic data, can aid in improving patient stratification and biomarker characterisation, identification of drug targets and designing personalised therapies in the context of oncology and beyond.

Reference

1. Stopfer LE, Flower CT, Gajadhar AS, et al. High-Density, Targeted Monitoring of Tyrosine Phosphorylation Reveals Activated Signaling Networks in Human Tumors. Cancer Research 2021; 81:2495–509. doi 1158/0008-5472.CAN-20-3804.