MyeloTrak: a sequencing-independent mass spectrometry assay for blood-based residual M-protein monitoring in multiple myeloma

With therapeutic advances in multiple myeloma, high-sensitivity disease monitoring has become crucial when the disease burden falls below the detection limit of conventional assays. While bone marrow (BM)-based minimal residual disease assessment is the gold standard, its invasiveness restricts the feasibility for frequent longitudinal monitoring. Bottom-up proteomics offers a non-invasive and highly sensitive alternative by detecting patient-specific clonotypic peptides in peripheral blood, but most approaches require BM RNA or full-length antibody sequencing to identify the clonotypic peptides. Hence, we developed the MyeloTrak assay, a sequencing-independent approach that identifies clonotypic peptides from plasma by leveraging intrinsic biological signatures of M-proteins. Monoclonal antibody-spiked samples and a patient cohort were utilized to validate the selection of clonotypic peptides benchmarked against known antibody and BM RNA sequences. Clonotypic peptides were enriched approximately 1,000-fold from a polyclonal background, enabling confident identification in 23 patients across diverse M-protein levels and isotypes. Selected clonotypic peptides remained detectable (idotp ≥ 0.9) at concentrations of 1–4 mg/L in serial dilution experiments using monoclonal antibody-spiked samples. Longitudinal tracking in 19 patients showed generally concordant trends with conventional assays, although four patients exhibited discordant trajectories. MyeloTrak supports the feasibility of identifying patient-specific clonotypic peptides directly from plasma by leveraging the inherent biological signatures of M-proteins. Because its discovery workflow does not require BM RNA or full-length immunoglobulin sequencing, it significantly reduces the complexity of blood-based residual M-protein monitoring.