{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,4,30]],"date-time":"2026-04-30T10:12:08Z","timestamp":1777543928693,"version":"3.51.4"},"reference-count":26,"publisher":"Walter de Gruyter GmbH","issue":"4","funder":[{"name":"National Science Foundation, Division of Materials Research","award":["DMR-1644779"],"award-info":[{"award-number":["DMR-1644779"]}]},{"name":"National Institutes of Health, National Cancer Institute","award":["CCSG P30 CA060553"],"award-info":[{"award-number":["CCSG P30 CA060553"]}]},{"name":"National Institutes of Health, National Institute of General Medical Sciences","award":["P41GM108569"],"award-info":[{"award-number":["P41GM108569"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2021,3,26]]},"abstract":"<jats:title>Abstract<\/jats:title>\n               <jats:sec id=\"j_cclm-2020-1072_abs_001\">\n                  <jats:title>Objectives<\/jats:title>\n                  <jats:p>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.<\/jats:p>\n               <\/jats:sec>\n               <jats:sec id=\"j_cclm-2020-1072_abs_002\">\n                  <jats:title>Methods<\/jats:title>\n                  <jats:p>Immunoprecipitation immunofixation (IP-IFE) was performed on a Sebia Hydrasys Scan2. Middle-down <jats:italic>de novo<\/jats:italic> 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.<\/jats:p>\n               <\/jats:sec>\n               <jats:sec id=\"j_cclm-2020-1072_abs_003\">\n                  <jats:title>Results<\/jats:title>\n                  <jats:p>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 <jats:italic>de novo<\/jats:italic> 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.<\/jats:p>\n               <\/jats:sec>\n               <jats:sec id=\"j_cclm-2020-1072_abs_004\">\n                  <jats:title>Conclusions<\/jats:title>\n                  <jats:p>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.<\/jats:p>\n               <\/jats:sec>","DOI":"10.1515\/cclm-2020-1072","type":"journal-article","created":{"date-parts":[[2020,10,24]],"date-time":"2020-10-24T05:37:41Z","timestamp":1603517861000},"page":"653-661","source":"Crossref","is-referenced-by-count":7,"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 <i>de novo<\/i> sequencing"],"prefix":"10.1515","volume":"59","author":[{"given":"W. Ian","family":"Deighan","sequence":"first","affiliation":[{"name":"Department of Clinical Chemistry , Altnagelvin Area Hospital , Londonderry , UK"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9178-2252","authenticated-orcid":false,"given":"Valerie J.","family":"Winton","sequence":"additional","affiliation":[{"name":"Proteomics Center of Excellence , Northwestern University , Evanston , IL , USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0349-235X","authenticated-orcid":false,"given":"Rafael D.","family":"Melani","sequence":"additional","affiliation":[{"name":"Proteomics Center of Excellence , Northwestern University , Evanston , IL , USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8633-0251","authenticated-orcid":false,"given":"Lissa C.","family":"Anderson","sequence":"additional","affiliation":[{"name":"Ion Cyclotron Resonance Program , National High Magnetic Field Laboratory , Tallahassee , FL , USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0527-8904","authenticated-orcid":false,"given":"John P.","family":"McGee","sequence":"additional","affiliation":[{"name":"Chemical and Biological Engineering , Northwestern University , Evanston , IL , USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6157-0937","authenticated-orcid":false,"given":"Luis F.","family":"Schachner","sequence":"additional","affiliation":[{"name":"Department of Chemistry , Northwestern University , Evanston , IL , USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"David","family":"Barnidge","sequence":"additional","affiliation":[{"name":"Laboratory Medicine and Pathology , Mayo Clinic College of Medicine , Rochester , MN , USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"David","family":"Murray","sequence":"additional","affiliation":[{"name":"Laboratory Medicine and Pathology , Mayo Clinic College of Medicine , Rochester , MN , USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"H. Denis","family":"Alexander","sequence":"additional","affiliation":[{"name":"Northern Ireland Centre for Stratified Medicine, Biomedical Sciences Research Institute , Ulster University , Londonderry , UK"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"David S.","family":"Gibson","sequence":"additional","affiliation":[{"name":"Northern Ireland Centre for Stratified Medicine, Biomedical Sciences Research Institute , Ulster University , Londonderry , UK"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Michael J.","family":"Deery","sequence":"additional","affiliation":[{"name":"Cambridge Centre for Proteomics , University of Cambridge , Cambridge , UK"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Feargal P.","family":"McNicholl","sequence":"additional","affiliation":[{"name":"Department of Haematology , Altnagelvin Area Hospital , Londonderry , UK"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Joseph","family":"McLaughlin","sequence":"additional","affiliation":[{"name":"Northern Ireland Centre for Stratified Medicine, Biomedical Sciences Research Institute , Ulster University , Londonderry , UK"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Neil L.","family":"Kelleher","sequence":"additional","affiliation":[{"name":"Proteomics Center of Excellence & Departments of Chemistry and Molecular Biology, Northwestern University , Evanston , IL , USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2887-4765","authenticated-orcid":false,"given":"Paul M.","family":"Thomas","sequence":"additional","affiliation":[{"name":"Proteomics Center of Excellence & Departments of Chemistry and Molecular Biology, Northwestern University , Evanston , IL , USA"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"374","published-online":{"date-parts":[[2020,10,20]]},"reference":[{"key":"2022031413522366984_j_cclm-2020-1072_ref_001","doi-asserted-by":"crossref","unstructured":"Mills, JR, Kohlhagen, MC, Willrich, MA, Kourelis, T, Dispenzieri, A, Murray, DL. 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