Development and Preliminary Validation of a MALDI-TOF MS Assay Using KTI as an Internal Standard for Serum M-Protein...

Background and Objectives: Conventional assays for M-protein detection in multiple myeloma (MM), including serum immunofixation electrophoresis (sIFE) and serum free light-chain (sFLC) assays, have limitations in selected clinical settings. This pilot study aimed to develop and preliminarily validate a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS)-based workflow using Kunitz trypsin inhibitor (KTI) as an internal standard for patient-specific serum M-protein light-chain tracking, particularly in low-level post-treatment samples in which conventional assays may be negative or difficult to interpret. Materials and Methods: A total of 55 serum samples from 25 patients with MM were analyzed. Serum immunoglobulin light-chain species were enriched using mixed κ/λ affinity beads, followed by reduction, KTI-based calibration, and MALDI-TOF MS analysis. Quantitative performance was evaluated using purified IgG1 κ standards. Time-matched sIFE and sFLC ratio results were used for descriptive comparison. Results: After KTI-based calibration, patient-specific M-protein light-chain molecular masses could be consistently identified. The assay showed good linearity over the range of 0.20-10.00 μg/mL, with a calibration equation of y = 6.0228x + 0.1063 and an R² of 0.9961. The limit of detection and limit of quantification were 0.002 μg/mL and 0.008 μg/mL, respectively. Intra-day and inter-day precision were acceptable, and recovery ranged from 96.0% to 101.2%. In selected low-level or discordant samples, including cases with therapeutic interference, polyclonal background, or non-secretory disease, MALDI-TOF MS provided exploratory complementary monitoring information. Conclusions: This KTI-calibrated MALDI-TOF MS workflow showed preliminary analytical performance within the validated low-concentration range and may serve as a complementary approach for patient-specific serum M-protein light-chain monitoring in selected clinical settings of MM. Larger independent studies are required before its clinical utility can be established.