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Immunoglobulins: expanding the role for mass spectrometry in protein biomarker quantification.

The impact of proteomics on clinical practice and laboratory medicine has been much anticipated, and many researchers have believed and predicted that the benefit would be primarily in the form of novel biomarker discovery. However, the most significant imminent improvement may be more straightforward: improved capability to measure clinical analytes and increased capacity for multiplexing to evaluate complex panels of disease biomarkers. Although discovery proteomics has revolutionized basic science in terms of defining protein complexes and molecular switching events determined by posttranslational modifications, the ongoing development of liquid chromatography-multiple reaction monitoring mass spectrometry (LC-MRM MS) (2) techniques and their application to protein biomarker quantification continues to improve the capability of the clinical laboratory.

Building on the long history of use in small-molecule quantification applied both to drugs and metabolites, LC-MRM MS has been used to monitor peptide surrogates for protein biomarkers of specific clinical interest such as apolipoprotein A-I (1), C-reactive protein (2), and prostate-specific antigen (3). Furthermore, peptide immunoprecipitation and mass spectrometry quantification have recently been applied to monitor thyroglobulin (4), overcoming obstacles associated with traditional antibody-based techniques. These advances can be applied to numerous other analytes relevant to human disease.

One such critically important and active area of research is the measurement of immunoglobulins, both endogenous and therapeutic. As an example, the impact of this research can be made immediately in multiple myeloma (MM), which is a tumor of the immunoglobulin-producing plasma cells. Clonal expansion of the tumor cells produces a monoclonal immunoglobulin, which can be quantified as a direct biomarker of disease burden. The clinical paradigm for evaluating patients relies on evaluation of the immunoglobulin in serum and urine by protein electrophoresis and immunofixation as well as quantification by nephelometry. Other assays (e.g., serum free light chains) can also be applied in combination with these techniques. Patients are typically assessed at 2- to 4-week intervals during treatment and 1- to 4-month intervals during remission. The immunoglobulin measurements are used in patient care to evaluate disease severity, monitor response to therapy, determine when to discontinue chemotherapy, and detect disease relapse. Improvements of these approaches could be expected to significantly impact the ability to define complete responses to chemotherapy, potentially eliminate minimal residual disease (MRD), and provide earlier detection of disease relapse, opening an earlier window for patient treatment. All of these aspects could enhance the ability to treat MM patients and improve their outcomes.

A method for quantification of immunoglobulins using peptides derived from tryptic digestion of the constant regions was proposed by our research team at the Moffitt Cancer Center as part of a review article on the role of quantitative proteomics in developing personalized care for cancer patients (5). 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. However, as the portfolio of clinical LC-MRM MS assays increases, this approach could become useful for implementation in the clinic.

Researchers at the Mayo Clinic have been working on the same problem of monitoring immunoglobulins in different disease settings, including MM, and have produced data for the feasibility and implementation of multiple mass spectrometry-based assays. These researchers have developed methods quantifying light chains using electrospray quadrupole-time-of-flight mass spectrometry, which provides a rapid analysis with improved sensitivity and molecular specificity (due to the measurement of the intact molecular weight) compared to protein electrophoresis (7). In the most recent investigation, reported in this issue of Clinical Chemistry, Ladwig et al. have evaluated quantification of IgG subclasses and compared the results to isoform-specific nephelometry in the context of immune deficiency and IgG4-related disease (8). Both this and the earlier publications illustrate methods that can be readily applied to the automated analysis of clinical samples. Their thorough and systematic approaches to testing these assays with clinical samples set a high standard and consistently illustrate the utility of quantitative mass spectrometry for assessment of protein biomarkers.

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. These methods will enter a very competitive space and must be compared to multiparameter flow cytometry (13) and genomic methods (14). However, retention of the current clinical paradigm of monitoring monoclonal immunoglobulin expression in serum has 2 benefits over genomic approaches for MRD detection using flow cytometry, allele-specific oligonucleotide PCR, or deep sequencing in serial bone marrow samples: lesser patient burden and systemic evaluation of disease. I fully expect that these methods will prove to have significant clinical value in MM. Regardless of how LC-MRM MS competes in this specific instance, the future is bright for quantitative proteomics to play a broader role in patient assessment as part of the clinical laboratory.

Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following 3 requirements: (a) significant contributions to the conception and design, acquisition of data, oranalysis and interpretation of data; (b) drafting or revising the article for intellectual content; and (c) final approval of the published article.

Authors' Disclosures or Potential Conflicts of Interest: Upon manuscript submission, all authors completed the author disclosure form.

Disclosures and/or potential conflicts of interest:

Employment or Leadership: None declared.

Consultant or Advisory Role: None declared.

Stock Ownership: None declared.

Honoraria: None declared.

Research Funding: J.M. Koomen, National Cancer Institute (R21 CA141285) and DeBartolo Family Personalized Medicine Institute Pilot Research Award in Personalized Medicine.

Expert Testimony: None declared.

Patents: J.M. Koomen. patent number 61946629.


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(4.) Hoofnagle AN, Becker JO, Wener MH, Heinecke JW. Quantification of thyroglobulin, a low-abundance serum protein, by immunoaffinity peptide enrichment and tandem mass spectrometry. Clin Chem 2008;54:1796-804.

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(6.) Remily-Wood ER, Benson K, Baz RC, Chen YA, Hussein M, Hartley-Brown MA, et al. Quantification of peptides from immunoglobulin constant and variable regions by liquid chromatography-multiple reaction monitoring mass spectrometry for assessment of multiple myeloma patients. Proteomics Clin Appl [Epub ahead of print 2014 Apr 10].

(7.) Barnidge DR, Dasari S, Botz CM, Murray DH, Snyder MR, Katzmann JA, et al. Using mass spectrometry to monitor monoclonal immunoglobulins in patients with a monoclonal gammopathy. J Proteome Res 2014;13:1419-27.

(8.) Ladwig PM, Barnidge DR, Snyder MR, Katzmann JA, Murray DL. Quantification of serum IgG subclasses by use of subclass-specific tryptic peptides and liquid chromatography-tandem mass spectrometry. Clin Chem 2014;60: 1080-8.

(9.) Barnidge DR, Tschumper RC, Theis JD, Snyder MR, Jelinek DF, Katzmann JA, et al. Monitoring M-proteins in patients with multiple myeloma using heavy-chain variable region clonotypic peptides and LC-MS/MS. J Proteome Res 2014;13:1905-10.

(10.) Evans VC, Barker G, Heesom KJ, Fan J, Bessant C, Matthews DA. De novo derivation of proteomes from transcriptomes for transcript and protein identification. Nat Methods 2012;9:1207-11.

(11.) Dekker LJ, Zeneyedpour L, Brouwer E, van Duijn MM, Sillevis Smitt PA, Luider TM. An antibody-based biomarker discovery method by mass spectrometry sequencing of complementarity determining regions. Anal Bioanal Chem 2011;399:1081-91.

(12.) Cheung WC, Beausoleil SA, Zhang X, Sato S, Schieferl SM, Wieler JS, et al. A proteomics approach for the identification and cloning of monoclonal antibodies from serum. Nat Biotechnol 2012;30:447-52.

(13.) Paiva B, Gutierrez NC, Rosinol L, Vidriales MB, Montalban MA, Martinez-Lopez J, et al. High-risk cytogenetics and persistent minimal residual disease by multiparameter flow cytometry predict unsustained complete response after autologous stem cell transplantation in multiple myeloma. Blood 2012;119:687-91.

(14.) Martinez-Lopez J, Lahuerta JJ, Pepin F, Gonzalez M, Barrio S, Ayala R, et al. Prognostic value of deep sequencing method for minimal residual disease detection in multiple myeloma. Blood 2014;123:3073-9.

John M. Koomen [1] *

[1] Molecular Oncology/Chemical Biology and Molecular Medicine, Moffitt Cancer Center, Tampa, FL.

* Address correspondence to this author at: Oncology/Chemical Biology and Molecular Medicine, Moffitt Cancer Center, 12902 Magnolia Drive, Tampa, FL 33612. Fax 813-745-3829; e-mail

Received May 20, 2014; accepted May 22, 2014.

Previously published online at DOI: 10.1373/Clinchem.2014.226035

[2] Nonstandard abbreviations: LC-MRM MS, liquid chromatography-multiple reaction monitoring mass spectrometry; MM, multiple myeloma; MRD, minimal residual disease.
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Title Annotation:Editorials
Author:Koomen, John M.
Publication:Clinical Chemistry
Article Type:Report
Date:Aug 1, 2014
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