Quadrupole mass spectrometry (LC-MS/MS) has long established itself as the workhorse of bioanalysis for small molecules, valued for its high sensitivity, selectivity, and robustness in regulated environments. However, its utility extends beyond pharmacokinetics and into the realm of mechanistic biomarker analysis. Here, we discuss our approach to measuring post-translational modification (PTM) of proteins as mechanistic biomarkers of target engagement.
Phosphorylation plays a central role in cellular signalling, often serving as a direct readout of pathway activation or inhibition. For drug programs targeting kinases, phosphatases, or associated signalling pathways, targeted phosphoproteomics offers a way to quantitatively assess pharmacodynamic effects. Rather than inferring activity through gene expression or downstream cytokine profiles, LC-MS/MS enables direct measurement of pathway-relevant phospho-sites on key proteins.
At Synexa, we have developed a simple, customisable workflow that begins with a discussion between our multi-disciplinary, scientific strategies team and our customer. We discuss target selection based on the chemistry, biology and mechanism of action of the potential drug in development. The generic workflow we would follow to establish and validate an assay for its context of use, whether as a clinical biomarker or in non-clinical, translational settings is summarised below.
Stage 1: In Silico Feasibility
Using in silico tools we identify potential signature peptides specific to the protein of interest employing a selection of enzymes that cleave the molecule at certain amino acids including trypsin, chymotrypsin, Glu-C and combinations if required.
Candidate peptides are evaluated based on sequence uniqueness, amenability to mass spectrometry (length, charge, and hydrophobicity), and the presence of known or putative phosphorylation sites. Priority is given to phosphopeptides that are functionally relevant (e.g., involved in activation loops or regulatory domains), conserved across species (for preclinical translation), and likely to yield robust MS signals.
Stage 2: Method Development
Once the target has been identified and the potential signature peptides have been selected, the feasibility experiments can begin. Initial digestions are performed on recombinant or purified protein standards initially in the absence of a biological matrix to optimise conditions without interference.
The selected protein is digested with the chosen enzyme and the signature peptides are identified using LC-MS/MS. Chromatographic conditions are optimised using a UPLC system coupled to the mass spectrometer for separation of the peptides. Signature peptides are screened for selectivity, sensitivity and stability and the digestion process is optimised.
The MS acquisition files are set up to detect both native and phosphorylated versions of the signature peptides where appropriate.
Further experiments are carried out in the appropriate biological matrix. This can include peripheral blood mononuclear cells (PMBCs), plasma, serum and others. Extraction and digestion methods are tested in the biological matrix. Samples may require pre-treatment prior to digestion and will require a post digestion cleanup prior to injection on the mass spectrometer. This may be solid phase extraction (SPE) or immunoprecipitation depending on the level of sensitivity and selectivity required.
Throughout development, peptide stability, matrix effects, and sample preparation recovery are assessed to ensure robust performance across replicates and batches.
Stage 3: Assay Qualification and Validation
If proof-of-concept data supports further development, the method can be qualified or validated for fit-for-purpose use, depending on regulatory needs. Reference standards of the peptides are custom synthesised with and without phosphorylation to allow for accurate calibration. Stable labelled versions are also prepared for use as internal standards.
The biomarker assay is validated to regulatory guidelines using a clinically relevant calibration range and QC samples. A suitable surrogate matrix is used to prepare the calibration standards.
Additional considerations: Proof of Concept or Confirmation of Biology
In some settings, our customers not only need a robust method established, but they also seek biological confirmation that their therapeutic candidate modulates the intended pathway. These non-clinical, exploratory experiments can provide early evidence of efficacy and help de-risk downstream development.
We can design stimulation assays in relevant primary cells or cell lines to assess whether the investigational compound promotes or inhibits phosphorylation at the site(s) of interest. This is typically done by comparing phosphorylated peptide levels across untreated, stimulated, and drug-treated conditions.
Such studies serve two purposes:
- Confirming Mechanism of Action – Demonstrating that the compound engages its target and modulates signalling as predicted.
- Identifying PD Markers – Establishing phosphopeptides that can serve as pharmacodynamic biomarkers in future in vivo or clinical studies.
These experiments can also help guide dose selection, time-course dynamics, and pathway cross-talk analysis. Importantly, they generate biologically relevant data without requiring animal studies or large sample volumes, making them well-suited for early development decision-making.
Synexa Platforms
At Synexa, our UK site is equipped with state-of-the-art UPLC-MS/MS platforms, ideally positioned just 15 minutes from Manchester Airport to support rapid turnaround for clinical studies. Operating from a GLP/GCP-accredited facility, we provide seamless bioanalytical and biomarker support throughout the entire drug development continuum – from pre-clinical to clinical phases.
Beyond LC-MS/MS, our comprehensive analytical capabilities, including ligand binding and flow cytometry assays, enable in-depth insights into post-translational modifications and other complex biological processes.
If you would like to learn more about how we can support your bioanalysis or biomarker needs, reach out to us at contactus@synexagroup.com.
