Quantification of peptides from immunoglobulin constant and variable regions by LC‐MRM MS for assessment of multiple myeloma patients

Abstract

Purpose Quantitative mass spectrometry assays for immunoglobulins (Igs) are compared with existing clinical methods in samples from patients with plasma cell dyscrasias, e.g. multiple myeloma. Experimental design Using LC-MS/MS data, Ig constant region peptides and transitions were selected for liquid chromatography-multiple reaction monitoring mass spectrometry (LC-MRM). Quantitative assays were used to assess Igs in serum from 83 patients. Results LC-MRM assays quantify serum levels of Igs and their isoforms (IgG1–4, IgA1–2, IgM, IgD, and IgE, as well as kappa(κ) and lambda(λ) light chains). LC-MRM quantification has been applied to single samples from a patient cohort and a longitudinal study of an IgE patient undergoing treatment, to enable comparison with existing clinical methods. Proof-of-concept data for defining and monitoring variable region peptides are provided using the H929 multiple myeloma cell line and two MM patients. Conclusions and Clinical Relevance LC-MRM assays targeting constant region peptides determine the type and isoform of the involved immunoglobulin and quantify its expression; the LC-MRM approach has improved sensitivity compared with the current clinical method, but slightly higher interassay variability. Detection of variable region peptides is a promising way to improve Ig quantification, which could produce a dramatic increase in sensitivity over existing methods, and could further complement current clinical techniques.

Quantitative analysis of immunoglobulin subclasses and subclass specific glycosylation by LC–MS–MRM in liver disease

“…One unique peptide was selected from each immunoglobulin for quantification of the subclasses [28]. Uniqueness of each selected peptide was confirmed by searching against UniProtKB/Swiss-Prot human protein database.…”

“…Uniqueness of each selected peptide was confirmed by searching against UniProtKB/Swiss-Prot human protein database. The corresponding isotopically labeled peptides or peptides with single conservative amino acid replacements were synthesized by Dr. Koomen’s lab [28;29] except for the IgG4 standard, which was synthesized by New England Peptide (Gardner, MA). These labeled peptides served as internal standards for quantification of immunoglobulins.…”

Standardisation of minimal residual disease in multiple myeloma

“…Run on peripheral blood, mass spectrometry is a tool able to discern the elements of a sample at a molecular and atomic sub-level, taking advantage of the differences of their mass-to-charge ratio. They considered that clonotypic peptides at the complementarity determining region (CDR) of the M protein, measured on serum (<1 μl), could be an ideal tool for MRD assessment in MM, that did not require DNA sequence information, and also was able to detect patchy or focal disease, that often the marrow aspiration cannot reveal, or in patients who were found negative by SPEP and IFE (Barnidge, Dasari, et al, 2014;Barnidge, Tschumper, et al, 2014;Dekker et al, 2011;Remily-Wood et al, 2014 Some studies used this to analyse the dynamic changes of serum proteomic spectra before and after chemotherapy.…”

Immunoglobulins: Expanding the Role for Mass Spectrometry in Protein Biomarker Quantification

“…This method parallels the nephelometry measurements of the total immunoglobulin concentrations (e.g., IgG, IgA, and IgM) with slightly improved sensitivity and a trade-off in precision (6). Our view was that the impact of changing the platform for this measurement from protein electrophoresis to mass spectrometry may not initially be great, because the measurements were parallel to current clinical techniques.…”

“…Although all of the methods described above have been analogous to current clinical assays, both groups have also worked in parallel on disease-specific immunoglobulin quantification using peptides from the variable region of the monoclonal immunoglobulin secreted by MM tumor cells (6, 9). On the basis of existing literature describing proteomics experiments informed by RNA sequencing (10) and analysis of therapeutic antibodies (11, 12), proof-of-concept experiments have been performed to assess the utility of this disease-specific peptide-based approach to monitor the monoclonal immunoglobulin in serum.…”