Multiple myeloma (MM) is a malignant plasma cell neoplasm, requiring the integration of clinical examination, laboratory and radiological investigations for diagnosis. Detection and isotypic identification of the monoclonal protein(s) and measurement of other relevant biomarkers in serum and urine are pivotal analyses. However, occasionally this approach fails to characterize complex protein signatures. Here we describe the development and application of next generation mass spectrometry (MS) techniques, and a novel adaptation of immunofixation, to interrogate non-canonical monoclonal immunoproteins. Immunoprecipitation immunofixation (IP-IFE) was performed on a Sebia Hydrasys Scan2. Middle-down de novo sequencing and native MS were performed with multiple instruments (21T FT-ICR, Q Exactive HF, Orbitrap Fusion Lumos, and Orbitrap Eclipse). Post-acquisition data analysis was performed using Xcalibur Qual Browser, ProSight Lite, and TDValidator. We adapted a novel variation of immunofixation electrophoresis (IFE) with an antibody-specific immunosubtraction step, providing insight into the clonal signature of gamma-zone monoclonal immunoglobulin (M-protein) species. We developed and applied advanced mass spectrometric techniques such as middle-down de novo sequencing to attain in-depth characterization of the primary sequence of an M-protein. Quaternary structures of M-proteins were elucidated by native MS, revealing a previously unprecedented non-covalently associated hetero-tetrameric immunoglobulin. Next generation proteomic solutions offer great potential for characterizing complex protein structures and may eventually replace current electrophoretic approaches for the identification and quantification of M-proteins. They can also contribute to greater understanding of MM pathogenesis, enabling classification of patients into new subtypes, improved risk stratification and the potential to inform decisions on future personalized treatment modalities.
Bibliographical noteFunding Information:
Research funding: National Science Foundation, Directorate for Mathematical and Physical Sciences, Division of Materials Research, DMR-1644779. U.S. Department of Health and Human Services, National Institutes of Health, National Cancer Institute, CCSG P30 CA060553. U.S. Department of Health and Human Services, National Institutes of Health, National Institute of General Medical Sciences, P41GM108569.
We thank our collaborators at Thermo Scientific who assisted with data acquisition on the Orbitrap Eclipse MS, including Kristina Srzentić, Romain Huguet, Christopher Mullen, and Philip Remes. Research reported in this publication was supported by the National Institute of General Medical Sciences of the National Institutes of Health under Award Number P41 GM108569 and by the National Cancer Institute CCSG P30 CA060553 awarded to the Robert H Lurie Comprehensive Cancer Center. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. A portion of this work was performed at the National High Magnetic Field Laboratory, which is supported by the National Science Foundation Cooperative Agreement No. DMR-1644779 and the State of Florida. LFS is a Gilliam Fellow of the Howard Hughes Medical Institute.
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- immunofixation electrophoresis
- multiple myeloma
- native mass spectrometry
- top-down protein sequencing
- truncated heavy chains