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Abstract INTRODUCTION:
M-protein is a key biomarker for the diagnosis and monitoring of multiple myeloma (MM). Conventional methods, including serum protein electrophoresis (SPEP), immunofixation, and free light chain assays, are limited in sensitivity and may not reliably detect disease at minimal (measurable) residual disease (MRD) levels. Mass spectrometry (MS)-based approaches have emerged as more sensitive alternatives. In this study, we implemented M-inSight (Sebia, Lisses, France), a clonotypic peptide-based assay, and evaluated its analytical performance for M-protein monitoring in patients with MM.
METHODS:
The M-inSight workflow consists of two sequential steps: (1) patient-specific clonotypic peptide identification and (2) longitudinal quantification.
For baseline characterization, serum samples underwent reduction, alkylation, and proteolytic digestion using multiple enzymes to maximize immunoglobulin sequence coverage. The resulting peptides were analyzed by nano-UPLC–MS/MS (Vanquish Neo UPLC system coupled with Orbitrap Exploris 480, Thermo Fisher Scientific, Waltham, MA, USA), and de novo sequencing was performed using the PeptID bioinformatics platform (Sebia). Clonotypic peptides derived from the complementarity-determining regions (CDRs) of both heavy and light chains were identified and defined as patient-specific signatures.
For longitudinal quantification, 2 μL of serum was processed using a standardized workflow, including spiking with stable isotope-labeled human IgG-kappa (SILuMAB, Sigma-Aldrich, St.Louis, MO, USA) as internal standard (IS), followed by reduction, alkylation, and tryptic digestion. Quantification of clonotypic peptides was performed using a parallel reaction monitoring (PRM) approach on a Vanquish Neo UPLC system coupled to an Orbitrap Exploris 120 mass spectrometer (Thermo Fisher Scientific), enabling sensitive and reproducible monitoring of patient-specific M-proteins over time.
Analytical performance was evaluated using a reference IgG1 kappa standard (RM8671, NIST, Gaithersburg, MD, USA) and a serum sample from a patient with MM (GCLabs-P01). Linearity, lower limit of quantification (LLOQ), and precision (intra- and inter-assay) were assessed. Serial dilutions were prepared in a single-donor serum matrix across a wide concentration range. Linearity was evaluated across the dilution series, and the LLOQ was defined as the lowest concentration meeting predefined accuracy and precision criteria. Precision was evaluated at high, medium, and low concentration levels. Intra-assay precision was determined using ten consecutive replicates per concentration, while inter-assay precision was assessed using three independent digestion preparations performed on different days, with five replicate analyses per preparation. Inter-laboratory reproducibility was evaluated by analyzing serial dilutions of RM8671 across two independent laboratories.
To evaluate concordance with conventional electrophoretic methods, serum samples from MM patients with longitudinal electrophoresis (EP) follow-up were analyzed using M-inSight. Patient-specific clonotypic peptides were identified, and subsequent samples were quantified accordingly. Results were compared with corresponding EP measurements to assess concordance.
RESULTS:
Excellent linearity was demonstrated across approximately five orders of magnitude, with coefficients of determination (R²) of 0.9991 for RM8671 and 0.997 for GCLabs-P01. The LLOQ was determined to be 0.001 g/L for RM8671 and 0.0022 g/L for GCLabs-P01.
Intra-assay precision showed coefficients of variation (CVs) below 10%, while inter-assay precision showed CVs below 15% across all tested concentration levels. Inter-laboratory comparison between GCLabs and Sebia Global demonstrated strong agreement, with R² values exceeding 0.90.
CDR-derived clonotypic peptide sequencing was successfully completed for all patients included in the EP comparison cohort, with at least four unique peptides identified per patient. Quantitative results obtained by M-inSight showed strong correlation with corresponding EP measurements. Notably, M-inSight enabled reliable monitoring in cases where M-protein was not detectable by EP, demonstrating superior sensitivity for low-level disease detection.
CONCLUSION:
M-inSight demonstrated a wide dynamic range and excellent analytical sensitivity for M-protein quantification. Although the LLOQ varied depending on patient-specific clonotypic peptides, the assay achieved an approximately 1,000-fold improvement in sensitivity compared with conventional SPEP (LLOQ ~1 g/L for SPEP vs. ~0.001 g/L for M-inSight). The method showed high reproducibility both within and across laboratories. In addition to strong concordance with electrophoretic methods, M-inSight enabled reliable detection and monitoring of M-protein below the EP detection limit. These findings highlight its ability to provide sensitive, patient-specific monitoring of disease burden. Furthermore, the assay supports minimally invasive, blood-based longitudinal monitoring and may enable earlier detection of disease changes, with potential clinical utility in treatment response assessment and relapse monitoring. Overall, M-inSight represents a robust and highly sensitive approach for M-protein quantification, with promising applications in low-level disease detection and MRD-level monitoring in MM. |
= Discovery stage. (57.21%, 2026)
= Translation stage. (23.38%, 2026)
= Clinically available. (19.40%, 2026)
