Ann Lab Med.  2024 Nov;44(6):518-528. 10.3343/alm.2024.0039.

Detecting M-Protein via Mass Spectrometry and Affinity Beads: Enrichment With Mixed Kappa-Lambda Beads Enables Prompt Application in Clinical Laboratories

• Lee J
• Choi JH
• Kim EH
• Im J
• Hwang H
• Yang S
• Lee JH
• Lee K
• Song J
• Park S
• Song SH

• KMID: 2560798
• DOI: http://doi.org/10.3343/alm.2024.0039

Abstract

Background
Detecting monoclonal protein (M-protein), a hallmark of plasma cell disorders, traditionally relies on methods such as protein electrophoresis, immune-electrophoresis, and immunofixation electrophoresis (IFE). Mass spectrometry (MS)-based methods, such as matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) and electrospray ionization-quadrupole time-of-flight (ESI-qTOF) MS, have emerged as sensitive methods. We explored the M-protein-detection efficacies of different MS techniques.
Methods
To isolate immunoglobulin and light chain proteins, six types of beads (IgG, IgA, IgM, kappa, lambda, and mixed kappa and lambda) were used to prepare samples along with CaptureSelect nanobody affinity beads (NBs). After purification, both MALDI-TOF MS and liquid chromatography coupled with Synapt G2 ESI-qTOF high-resolution MS analysis were performed. We purified 25 normal and 25 abnormal IFE samples using NBs and MALDI-TOF MS (NB-MALDI-TOF).
Results
Abnormal samples showed monoclonal peaks, whereas normal samples showed polyclonal peaks. The IgG and mixed kappa and lambda beads showed monoclonal peaks following the use of daratumumab (an IgG/kappa type of monoclonal antibody) with both MALDI-TOF and ESI-qTOF MS analysis. The limits of detection for MALDI-TOF MS and ESIqTOF MS were established as 0.1 g/dL and 0.025 g/dL, respectively. NB-MALDI-TOF and IFE exhibited comparable sensitivity and specificity (92% and 92%, respectively).
Conclusions
NBs for M-protein detection, particularly with mixed kappa-lambda beads, identified monoclonal peaks with both MALDI-TOF and ESI-qTOF analyses. Qualitative analysis using MALDI-TOF yielded results comparable with that of IFE. NB-MALDI-TOF might be used as an alternative method to replace conventional tests (such as IFE) to detect M-protein with high sensitivity.

Keyword

Immunofixation electrophoresis; MALDI-TOF; Mass spectrometry; Monoclonal protein; Multiple myeloma; qTOF

Figure

• 

  Fig. 1 Negative results found with IFE (A) and NB-MALDI-TOF (B). The m/z ranges of single- and double-charged light chains are presented in the red boxes. The X-axis represents the mass-to-charge ratio (m/z), and the Y-axis shows the summed signal intensity for 500 shots using MALDI-TOF. Abbreviations: Intens., intensity; IgA, immunoglobulin A; IgG, immunoglobulin G; IFE, immunofixation electrophoresis; NB-MALDI-TOF, nanobody affinity beads combined with matrix-assisted laser desorption/ionization time-of-flight; K, kappa; KnL, mixed kappa and lambda beads; L, lambda; 1+ LC, single-charged light chain; 2+ LC, double-charged light chain; IgM, immunoglobulin M; m/z, mass-to-charge ratio.

• 

  Fig. 2 Examples of NB-MALDI-TOF spectra found with normal and abnormal samples. (A) Polyclonal peaks observed with IgG beads. (B) Polyclonal peaks observed with mixed kappa and lambda. (C) Monoclonal peaks observed with IgG beads. (D) Monoclonal peaks observed with an abnormal IFE sample identified as having the IgG/kappa subtype. Abbreviations: IFE, immunofixation electrophoresis; KnL, kappa and lambda mixed beads; 1+ LC, single-charged light chain; 2+ LC, double-charged light chain; NB-MALDI-TOF, nanobody affinity beads combined with matrix-assisted laser desorption/ionization time-of-flight.

• 

  Fig. 3 Evaluation of limit of detection of NB-MALDI-TOF method following IgG purification using normal serum sample (A) and samples spiked with daratumumab at concentrations of 0.05 (B), 0.1 (C), 0.2 (D), 0.5 (E), or 1.0 g/dL (F). Abbreviations: 1+ LC, single-charged light chain; 2+ LC, double-charged light chain; NB-MALDI-TOF, nanobody affinity beads combined with matrix-assisted laser desorption/ionization time-of-flight.

• 

  Fig. 4 NB-LC-ESI-qTOF results after purification with IgG, IgA, IgM, mixed kappa and lambda, kappa, and lambda beads. Mass spectra of samples after NB purification with IgG (A), IgA (B), IgM (C), KnL (D), kappa (E), and lambda (F) beads. Normal pooled serum spiked with daratumumab (0.1 g/dL) showed positive results with monoclonal peaks in the mass spectra after purification with IgG, KnL, and kappa beads (A, D, E) and negative results after purification with IgA, IgM, and lambda beads (B, C, F). The X-axis represents the mass values after deconvolution, and the Y-axis shows the relative intensity observed using NB-LC-ESI-qTOF. The intensity is annotated in the top right corner of each spectrum at 100% relative intensity. Abbreviations: KnL, mixed kappa and lambda; NB, nanobody affinity bead; NB-LC-ESI-qTOF, nanobody affinity beads combined with liquid chromatography-electrospray ionization-quadrupole time-of-flight.

Reference

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