bioanalysis
CASE STUDY
Expert Voice: Ashley Phillips
LC-MS for Protein Biomarkers: Precision Where Immunoassays Fall Short
Originally recorded January 2026 | Bioanalysis Zone
Meet Ashley Phillips
Ashley Phillips is a senior scientist in Resolian’s bioanalytical team, specialising in the development of LC-MS-based methods for complex proteins and biomarkers. His work sits at the intersection of method innovation and real-world drug development, solving problems that standard immunoassay platforms simply cannot address.
In an interview with Bioanalysis Zone, Ashley shared his thinking on when and why LC-MS is the right tool for protein biomarker measurement, illustrated with a detailed example from a recent assay development project.
Watch the interview: LC-MS for Protein Biomarkers: An Interview with Ashley Phillips
Why LC-MS for Proteins?
For many protein biomarker assays, ligand-binding assays (LBAs) remain the default. They are well-established, sensitive, and familiar to most development teams. But Ashley makes the case that LC-MS offers a distinct set of advantages that, in the right context, make it the more scientifically sound choice.
Selectivity without the reagent dependency. LC-MS uses mass-based specificity to identify a surrogate peptide that can only come from the target protein. It is not reliant on a matched antibody pair, which means it is more tolerant of lower-quality or less-specific capture reagents.
Isoform differentiation. Where immunoassays struggle to distinguish between closely related protein isoforms, LC-MS can select surrogate peptides unique to each, enabling precise, individual measurement.
Scalable sensitivity. At higher LLOQ requirements, a straightforward total digest approach can be used. When greater sensitivity is needed, an immunocapture step can be added to create a hybrid LC-MS approach. The design scales to the scientific requirement.
Drug-tolerant total assay design. Because an indiscriminate digest approach liberates the surrogate peptide regardless of molecular form, total analyte measurement is achievable even where the therapeutic shares structural similarity with the endogenous target.
“Because only a single capture reagent is required, rather than a matched pair of antibodies, the technique is also more tolerant of lower-quality antibodies.”
The Biomarker Advantage
The application of LC-MS to biomarker measurement carries a specific advantage that Ashley highlights: when a dosed therapeutic binds and captures the biomarker, the biomarker adopts the half-life of the therapeutic and its concentration rises. This brings it into the sensitivity range of protein LC-MS assays, opening up measurement windows that would otherwise be inaccessible.
At the same time, where the therapeutic is structurally similar to the endogenous biomarker, specific surrogate peptides can be selected to differentiate between them, ensuring accurate quantitation of the endogenous material without interference from the drug itself.
Hitting the Limits of Your Immunoassay Platform?
Resolian’s LC-MS team builds mass-based protein and biomarker assays that solve what standard immunoassays can’t, from isoform-specific quantitation to drug-tolerant total analyte measurement.
A Real-World Example: IgG Subtype Multiplexing
Ashley describes a recent assay developed to measure total IgG and all four IgG subtypes simultaneously from human serum. The client needed to track how a biotherapeutic designed to reduce IgG levels was performing across all subtypes.
The choice of LC-MS over immunoassay was straightforward: the therapeutic shared high structural similarity with the endogenous IgG, meaning an antibody-based approach would risk overestimating the endogenous levels. LC-MS, with subtype-specific surrogate peptides, could distinguish the two precisely.
The assay design was intentionally lean: no capture reagents, a high-temperature rapid tryptic digest, and five analytes monitored simultaneously, one surrogate peptide per subtype plus one shared peptide for total IgG.
Navigating the Development Challenges
Five analytes multiplexed into a single assay, spanning a dynamic range from above 12 mg/mL to below 1 mg/mL, created real complexity. Ashley explains how the team resolved it:
- Minimum required dilution (MRD) calibration: carefully selected so samples were diluted into the assay range without losing the ability to characterise the biotherapeutic’s mechanism of action.
- MS parameter optimisation: different parent and fragment ion transitions were selected per analyte. Highly sensitive transitions for lower-concentration subtypes such as IgG3 and IgG4; less sensitive transitions for higher-concentration analytes to prevent signal saturation.
- Design of experiments (DoE) for digestion: conditions were systematically optimised to maximise response for lower-concentration analytes while maintaining sufficient sensitivity across the full panel.
A further challenge arose during parallelism assessment, where the initial surrogate matrix could not demonstrate reproducible parallelism. A replacement matrix was required, subject to several constraints, and a structured screening exercise across multiple candidates identified a suitable option. Lot-to-lot consistency was then confirmed before validation.
“Following validation, the assay entered sample analysis, where it has processed a large number of samples with a very high pass rate, indicating strong assay performance.”
Knowing the Limits
Ashley is candid about where LC-MS has constraints. Sensitivity is the primary limitation: protein LC-MS is inherently less sensitive than ligand-binding assays, which can restrict its use where very low concentrations must be measured.
These assays are also more complex to develop. They often require bespoke designs, substantial scientific experience, and access to non-standard tools, whether that means automated immunocapture systems such as KingFisher, high-resolution mass spectrometry, 2D-LC, or microflow LC.
That complexity is also, of course, where the expertise lives. For sponsors working on therapeutics where standard immunoassay approaches are insufficient, Resolian’s depth in this area provides a scientifically rigorous alternative.
Watch the interview: Watch the full interview on Bioanalysis Zone
Ready to Talk Through Your Protein Biomarker Assay?
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