MSACL 2025 Abstract

Amino Acid Sequence-Based, Reagent Antibody-Free Isotyping of Serum M-Proteins Using High-Resolution Mass Spectrometry

Priscilla S.-W. Yeung (1, 2), Chenyin Lu (1), Yajing Liu (1), Ruben Y. Luo (1, 2)
(1) Department of Pathology, Stanford University, Stanford, CA, USA (2) Clinical Laboratories, Stanford Health Care, Palo Alto, CA, USA

Priscilla Yeung, MD, PhD (Presenter) Stanford/UCSF

Presenter Bio: Priscilla Yeung is a Fellow in the Joint Stanford-UCSF Clinical Chemistry Fellowship interested in using mass spectrometry to discover improved biomarkers for clinical testing. Her current research with Dr. Ruben Luo is focused on applying top-down mass spectrometry to the diagnosis of monoclonal gammopathies. Prior to residency, she completed her MD/PhD training at Northwestern University, where she studied the biophysical mechanisms of calcium channel gating with Dr. Murali Prakriya, and her undergraduate studies at University of Pennsylvania, where she studied amyloid-beta protein misfolding with Dr. Paul Axelsen.

Relevant Financial Disclosures (within past 24 months, reported on Apr 11, 2026) No relevant financial relationship(s) to disclose.

INTRODUCTION:
The determination of M-protein isotypes is an essential component of the clinical laboratory evaluation of plasma cell disorders, including multiple myeloma, plasmacytoma, and amyloidosis. In addition to its use in disease monitoring, M-protein isotypes also confer prognostic information for myeloma patients. M-protein isotyping is predominantly performed using gel electrophoresis immunofixation (IFE), capillary electrophoresis immunosubtraction, or MALDI-TOF (MASS-FIX). All three of these currently available methods utilize reagent antibodies that bind to specific epitopes to identify isotypes. By contrast, modern high-resolution mass spectrometry (HR-MS) is able to characterize the amino acid sequences of proteins through molecular dissociation techniques. In this study, we report a proof-of-concept methodology to determine the isotype of M-proteins solely based on HR-MS.

METHODS:
Remnant patient samples with serum IFE results from the Stanford Clinical Chemistry Laboratory were collected and processed according to Institutional Review Board protocols approved by Stanford Health Care. Diluted serum samples were digested with antibody heavy chain cleavage enzymes, reduced by TCEP, and processed using Melon Gel purification kits. M-Protein light chain and Fd (fragment of heavy chain containing the variable and CH1 regions) were extracted from serum using this workflow. HR-MS analysis was performed on a Vanquish Flex multi-channel HPLC coupled with a Q-Exactive Plus mass spectrometer or a Vanquish Neo HPLC coupled with an Orbitrap Eclipse Tribrid mass spectrometer. Proteins were separated on MAbPac reverse phase columns. Mobile phase A was 0.1% formic acid in water and mobile phase B was 0.1% formic acid in acetonitrile. MS2 fragmentation was performed using a combination of different molecular dissociation techniques.

RESULTS:
The performance of the HR-MS-based M-protein isotyping method was assessed by comparison with IFE results. For the isotyping of light chains and IgG heavy chains, this method demonstrated a ~95% concordance rate with IFE in over 100 patient samples. Discrepancies arose in cases of bi-clonal or heavily glycosylated M-proteins. Distinct glycosylation patterns were identified in the light chain and/or Fd regions of a fraction of IgG samples. The method to isotype IgA and IgM heavy chains is currently being optimized.

CONCLUSION:
This proof-of-concept study demonstrates the potential of a HR-MS-based method as an antibody-free approach to accurately determine the isotype and post-translational modifications of serum M-proteins.