MSACL 2026 Abstract

Grossly Intact: Comparing Internal Standards in a Multiplexed Protein LC-MS/MS Assay

Erin Toledano Strom (1), Matthew D. Gray (2), Jim Boonyaratanakornkit (2,3), Andy Hoofnagle (1), Rebecca S. Treger (1)
(1) Department of Laboratory Medicine and Pathology, University of Washington, Seattle, WA, (2) Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Center, Seattle, WA, (3) Department of Medicine, University of Washington, Seattle, WA

Rebecca Treger, MD, PhD (Presenter) University of Washington

Presenter Bio: I completed my undergraduate degree in Molecular, Cellular, & Developmental Biology at Yale University before pursuing a combined MD/PhD degree through the Medical Scientist Training Program at the Yale School of Medicine. I completed my PhD training in Immunobiology, investigating natural antibodies and innate immunity to transposable elements. My doctoral work included significant assay development, which solidified my clinical interest in the field of laboratory medicine. I therefore completed residency training in Clinical Pathology at the University of Washington, which I then joined as an Assistant Professor in 2023 to pursue an academic career focused on clinical immunology and method development in laboratory medicine. I currently serve as Medical Director of the University of Washington Clinical Immunology lab, and am also Co-Director of the University of Washington Post-Sophomore Fellowship Program.

Relevant Financial Disclosures (within past 24 months, reported on Apr 17, 2026) No relevant financial relationship(s) to disclose.

INTRODUCTION:
Clinical measurement of total immunoglobulin G (IgG) and IgG1-4 subclass antibodies supports the diagnosis and monitoring of plasma cell neoplasms and IgG4-related disease. IgG antibodies are typically quantified clinically using immunoassays, which are susceptible to antigen excess and may also fail to react with epitopes present on rare IgG allotypes. Additionally, commercially available IgG subclass immunoassays are calibrated using different reference standards, yielding significant inter-assay discrepancies in quantitation and reference intervals. Thus, quantitative measurement of IgG subclasses using a liquid chromatography tandem mass spectrometry (LC-MS/MS) method to detect highly conserved heavy chain sequences could successfully circumvent these immunoassay limitations. However, IgG heavy chains are glycosylated and connected by disulfide bonds, and the extent to which tryptic or cleavable peptide internal standardization can yield accurate heavy chain measurement is not known.

METHODS:
We generated glycosylated, full-length stable isotope-labeled recombinant IgG1-4 kappa internal standards (rIS) and compared them to stable isotope-labeled tryptic and winged cleavable peptide internal standards (tIS and wIS) for quantification of IgG1-4 in tryptic digests of human serum using LC-MS/MS on a triple quadrupole mass analyzer. Proteotypic peptide peak area ratios (PAR) were measured in triplicate on each of three days in two serum pools with elevated IgG1-3 and low IgG4 or elevated IgG4 and low IgG1-3. Internal standardization was carried out using: (1) equimolar rIS & wIS, (2) equimolar wIS & post-digestion tIS, (3) equimolar wIS & pre-digestion tIS, or (4) rIS & wIS & post-digestion tIS with non-equimolar rIS optimized to minimize interference with the IgG2 and IgG3 tIS to below 5%.

RESULTS:
We observed that the PAR obtained using rIS demonstrated overall superior precision compared to those obtained using wIS or tIS across all IgG subclasses and antibody concentrations, with total (root sum squared) coefficients of variation for the rIS ranging from 4.1-9.4%. IgG subclass wIS PAR were comparable to those obtained using either rIS or pre-digestion tIS, while the post-digestion addition of tIS resulted in 25-50% reductions in PAR with similar precision. Moreover, compared to the tIS added pre- or post-digestion, the wIS demonstrated worse PAR precision for all IgG subclasses at almost all antibody concentrations. However, most notably for the IgG1 wIS PAR, interday variability was higher than intraday variability, suggesting that external calibration with a single point calibrator may improve its reproducibility. Lastly, reduction of rIS IgG2 and IgG3 molar ratios to minimize interference with the tIS decreased the calculated IgG2 and IgG3 PAR but did not meaningfully impact precision, compared to samples containing equimolar quantities of rIS and wIS.

CONCLUSION:
Overall, our results indicate that rIS exhibit superior reproducibility compared to wIS and tIS for measurement of all IgG subclasses across concentrations in pooled human serum. Our findings also suggest that there is no clear benefit of using wIS in place of tIS added prior to digestion, which can successfully account for the subclass-dependent peptide degradation that occurs during sample preparation. Follow-up studies are ongoing to fully validate this multiplexed IgG subclass LC-MS/MS assay and compare rIS to wIS and pre-digestion tIS for internal standardization, alongside external calibration to reduce interday variability.