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

W. Ian Deighan; Valerie J. Winton; Rafael D. Melani; Lissa C. Anderson; John P. McGee; Luis F. Schachner; David Barnidge; David Murray; H. Denis Alexander; David S. Gibson; Michael J. Deery; Feargal P. McNicholl; Joseph McLaughlin; Neil L. Kelleher; Paul M. Thomas

doi:10.1515/cclm-2020-1072

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

W. Ian Deighan, Valerie J. Winton, Rafael D. Melani, Lissa C. Anderson, John P. McGee, Luis F. Schachner, David Barnidge, David Murray, H. Denis Alexander, David S. Gibson, Michael J. Deery, Feargal P. McNicholl, Joseph McLaughlin, Neil L. Kelleher, Paul M. Thomas

Research output: Contribution to journal › Article › peer-review

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Abstract

Objectives: 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.

Methods: 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.

Results: 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.

Conclusions: 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.

Original language	English
Pages (from-to)	653-661
Number of pages	9
Journal	Clinical Chemistry and Laboratory Medicine
Volume	59
Issue number	4
Early online date	20 Oct 2020
DOIs	https://doi.org/10.1515/cclm-2020-1072
Publication status	Published (in print/issue) - 26 Mar 2021

Bibliographical note

Funding 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.

Funding Information:
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.

Publisher Copyright:
© 2020 Walter de Gruyter GmbH, Berlin/Boston.

Copyright:
Copyright 2020 Elsevier B.V., All rights reserved.

Keywords

immunofixation electrophoresis
multiple myeloma
native mass spectrometry
top-down protein sequencing
truncated heavy chains
Myeloma Proteins
Humans
Mass Spectrometry
Proteomics/methods
Antibodies, Monoclonal
Immunoelectrophoresis
Multiple Myeloma/diagnosis

UN SDGs

This output contributes to the following UN Sustainable Development Goals (SDGs)

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Deighan, W. I., Winton, V. J., Melani, R. D., Anderson, L. C., McGee, J. P., Schachner, L. F., Barnidge, D., Murray, D., Alexander, H. D., Gibson, D. S., Deery, M. J., McNicholl, F. P., McLaughlin, J., Kelleher, N. L., & Thomas, P. M. (2021). 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. Clinical Chemistry and Laboratory Medicine, 59(4), 653-661. https://doi.org/10.1515/cclm-2020-1072

@article{5775696826c2448fbb3b95772a49b6d3,

title = "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 = "Objectives: 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.Methods: 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.Results: 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.Conclusions: 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.",

keywords = "immunofixation electrophoresis, multiple myeloma, native mass spectrometry, top-down protein sequencing, truncated heavy chains, Myeloma Proteins, Humans, Mass Spectrometry, Proteomics/methods, Antibodies, Monoclonal, Immunoelectrophoresis, Multiple Myeloma/diagnosis",

author = "Deighan, {W. Ian} and Winton, {Valerie J.} and Melani, {Rafael D.} and Anderson, {Lissa C.} and McGee, {John P.} and Schachner, {Luis F.} and David Barnidge and David Murray and Alexander, {H. Denis} and Gibson, {David S.} and Deery, {Michael J.} and McNicholl, {Feargal P.} and Joseph McLaughlin and Kelleher, {Neil L.} and Thomas, {Paul M.}",

note = "Funding 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. Funding Information: We thank our collaborators at Thermo Scientific who assisted with data acquisition on the Orbitrap Eclipse MS, including Kristina Srzenti{\'c}, 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. Publisher Copyright: {\textcopyright} 2020 Walter de Gruyter GmbH, Berlin/Boston. Copyright: Copyright 2020 Elsevier B.V., All rights reserved.",

year = "2021",

month = mar,

day = "26",

doi = "10.1515/cclm-2020-1072",

language = "English",

volume = "59",

pages = "653--661",

journal = "Clinical Chemistry and Laboratory Medicine",

issn = "1434-6621",

publisher = "De Gruyter",

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Deighan, WI, Winton, VJ, Melani, RD, Anderson, LC, McGee, JP, Schachner, LF, Barnidge, D, Murray, D, Alexander, HD, Gibson, DS, Deery, MJ, McNicholl, FP, McLaughlin, J, Kelleher, NL & Thomas, PM 2021, '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', Clinical Chemistry and Laboratory Medicine, vol. 59, no. 4, pp. 653-661. https://doi.org/10.1515/cclm-2020-1072

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. / Deighan, W. Ian; Winton, Valerie J.; Melani, Rafael D. et al.
In: Clinical Chemistry and Laboratory Medicine, Vol. 59, No. 4, 26.03.2021, p. 653-661.

Research output: Contribution to journal › Article › peer-review

TY - JOUR

T1 - 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

AU - Deighan, W. Ian

AU - Winton, Valerie J.

AU - Melani, Rafael D.

AU - Anderson, Lissa C.

AU - McGee, John P.

AU - Schachner, Luis F.

AU - Barnidge, David

AU - Murray, David

AU - Alexander, H. Denis

AU - Gibson, David S.

AU - Deery, Michael J.

AU - McNicholl, Feargal P.

AU - McLaughlin, Joseph

AU - Kelleher, Neil L.

AU - Thomas, Paul M.

N1 - Funding 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. Funding Information: 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. Publisher Copyright: © 2020 Walter de Gruyter GmbH, Berlin/Boston. Copyright: Copyright 2020 Elsevier B.V., All rights reserved.

PY - 2021/3/26

Y1 - 2021/3/26

N2 - Objectives: 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.Methods: 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.Results: 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.Conclusions: 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.

AB - Objectives: 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.Methods: 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.Results: 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.Conclusions: 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.

KW - immunofixation electrophoresis

KW - multiple myeloma

KW - native mass spectrometry

KW - top-down protein sequencing

KW - truncated heavy chains

KW - Myeloma Proteins

KW - Humans

KW - Mass Spectrometry

KW - Proteomics/methods

KW - Antibodies, Monoclonal

KW - Immunoelectrophoresis

KW - Multiple Myeloma/diagnosis

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DO - 10.1515/cclm-2020-1072

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SN - 1434-6621

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JO - Clinical Chemistry and Laboratory Medicine

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Deighan WI, Winton VJ, Melani RD, Anderson LC, McGee JP, Schachner LF et al. 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. Clinical Chemistry and Laboratory Medicine. 2021 Mar 26;59(4):653-661. Epub 2020 Oct 20. doi: 10.1515/cclm-2020-1072

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

Bibliographical note

Keywords

UN SDGs

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