Development of novel methods for non-canonical myeloma protein analysis with an innovative adaptation of immunofixation electrophoresis, native top-down mass spectrometry, and middle-down de novo sequencing

Abstract

ObjectivesMultiple 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.MethodsImmunoprecipitation 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.ResultsWe 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.ConclusionsNext 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.

Direct Mass Spectrometry-Based Detection and Antibody Sequencing of Monoclonal Gammopathy of Undetermined Significance from Patient Serum: A Case Study

“…While the Fab-profiling workflow presented here is limited to the IgG1 subclass, and the de novo sequencing and glycan profiling methods are best suited for research applications, we believe that a robust implementation of Fab profiling across all IgG subclasses by LC-MS holds promise for the clinical detection of M-proteins and diagnostics of monoclonal gammopathies. The outlined approach in this case study adds to an expanding mass spectrometry-based toolkit to characterize monoclonal gammopathies such as MGUS and MM with fine molecular detail. − The ability to detect monoclonal gammopathies and determine M-protein sequences straight from peripheral blood samples by mass spectrometry provides opportunities to understand the molecular mechanisms of these diseases.…”

Direct mass spectrometry-based detection and antibody sequencing of Monoclonal Gammopathy of Undetermined Significance from patient serum – a case study

“…The outlined approach adds to an expanding mass spectrometry-based toolkit to characterize monoclonal gammopathies such as MGUS and MM with fine molecular detail. [15][16][17][18][19][20] The ability to detect monoclonal gammopathies and determine Mprotein sequences straight from peripheral blood samples by mass spectrometry provides opportunities to understand the molecular mechanisms of these diseases.…”