Article pubs.acs.org/jpr
Monitoring M‑Proteins in Patients with Multiple Myeloma Using Heavy-Chain Variable Region Clonotypic Peptides and LC−MS/MS David R. Barnidge,† Renee C. Tschumper,‡ Jason D. Theis,† Melissa R. Snyder,† Diane F. Jelinek,‡ Jerry A. Katzmann,† Angela Dispenzieri,†,§ and David L. Murray*,† †
Department of Laboratory Medicine and Pathology, ‡Department of Immunology, §Department of Hematology, Mayo Clinic, Rochester, Minnesota 55905, United States S Supporting Information *
ABSTRACT: Multiple myeloma is a disease characterized by a clonal expansion of plasma cells that secrete a monoclonal immunoglobulin also referred to as an M-protein. In the clinical laboratory, protein electrophoresis (PEL), immunofixation electrophoresis (IFE), and free light chain nephelometry (FLC) are used to detect, monitor, and quantify an M-protein. Here, we present an alternative method based on monitoring a clonotypic (i.e., clonespecific) peptide from the M-protein heavy chain variable region using LC− MS/MS. Tryptic digests were performed on IgG purified serum from 10 patients with a known IgG M-protein. Digests were analyzed by shotgun LC− MS/MS, and the results were searched against a protein database with the patient specific, heavy chain variable region gene sequence added to the database. In all 10 cases, the protein database search matched multiple clonotypic peptides from each patient’s heavy chain variable region. The clonotypic peptides were then used to quantitate the amount of M-protein in patient serum samples using selected reaction monitoring (SRM) on a triple quadrupole mass spectrometer. The response for the clonotypic peptide observed by SRM correlated with the M-protein observed by PEL. In addition, the clonotypic peptide was clearly observed by SRM in samples that were negative by IFE and FLC. Monitoring clonotypic peptides using SRM has the capacity to redefine clinical residual disease because of its superior sensitivity and specificity compared with current analytical methods. KEYWORDS: multiple myeloma, mass spectrometry, peptide, variable region, clonotypic, M-protein, digest, specificity, individualized, biomarker, SRM, proteotypic
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INTRODUCTION Multiple myeloma (MM) is a malignancy characterized by a clonal bone marrow plasma cell expansion that manifests in end organ damage in the form of anemia, hypercalcemia, bone lesions, and renal impairment.1 When a clinical suspicion of MM exists, serum is tested for the presence of an M-protein using agarose protein electrophoresis (PEL) with Coomassie staining. If a peak from an M-protein, also referred to as an Mspike, is observed, additional immunofixation (IFE) is performed to determine the isotype of the M-protein.2 Most, but not all, neoplastic plasma cells produce an M-protein; however, some neoplastic plasma cells will not secrete intact immunoglobulins. Therefore, current guidelines also suggest measuring the free-light chain (FLC) ratio because abnormal FLC ratios can detect a significant proportion of these nonsecretory myelomas. In light of clinical symptoms and positive serum screening, a bone marrow biopsy is performed to verify the presence of plasma cells. The M-protein concentration in the majority of newly diagnosed MM patients is greater than 3 g/dL (200 μM) but shortly after treatment the M-protein concentration changes dramatically, oftentimes dropping orders of magnitude in a span of a few months as malignant clonal plasma cells are eliminated.3 Tracking the © 2014 American Chemical Society
concentration of the M-protein is considered one of the best ways to monitor a patient’s response to treatment because the M-protein is a sensitive and specific biomarker for MM and new more sensitive methods are needed to monitor the Mprotein.4 A new proteomic-based method for the detection of specific antibodies is beginning to emerge in the literature.5 This approach is fundamentally different from current clinical laboratory methods because it uses the primary sequence of the variable regions of immunoglobulins. On the basis of the widely accepted protein quantification technique in which a proteotypic tryptic peptide is used to quantify proteins by LC− MS/MS,6−9 a highly specific clonotypic tryptic peptide from the Ig variable region can be selected for quantitation. This approach to monitoring a monoclonal immunoglobulin has been described by others who have detected a tryptic peptide from the variable region of a recombinant mAb in serum from cynomolgus monkeys10 and the mAb adalimumab spiked into normal human serum.11 In this paper, we apply the same methodology to the M-protein in MM patients to result in an Received: October 8, 2013 Published: February 19, 2014 1905
dx.doi.org/10.1021/pr5000544 | J. Proteome Res. 2014, 13, 1905−1910
Journal of Proteome Research
Article
5600 Q-TOF mass spectrometer (ABSciex, Framingham, MA) run in positive ESI mode. A 2 μL volume of digest was injected onto an Eksigent 0.3 × 50 mm, EP-120, C8 column with 3 μm packing flowing at 10 μL/min. An aqueous (A: water + 0.1% formic acid) to organic (B: acetonitrile + 0.1% formic acid) gradient was used to separate the tryptic peptides within a 15 min run starting out at 95% A/5% B, held for 2 min, ramped to 60% A/40% B over 2 min, then ramped to 45% A/55% B over 5 min, then ramped to 5% A/95% B over 2 min, and held for 2 min before cycling back to 95% A/5% B. The mass spectrometer method was set to perform TOF-MS scans from 300 to 1600 m/z with a 250 ms accumulation time. The instrument was set to trigger an MS/MS scan on any +2, +3, +4, or +5 charged ion with counts per second abundance over 150. A total of 30 product ion MS/MS scans were allowed per TOF-MS scan. Each product ion had an accumulation time of 50 ms with a scan range from 100 to 2500 m/z. The Turbo V ion source was given the following settings: ISV, 5500; TEMP, 500; CUR, 45; GS1, 35; GS2, 30. The DP was set at 100 and the CE for MS/MS was set at 35 with a CES of 10. The instrument was calibrated using ABSciex API positive ion calibration solution infused into the APCI probe adjacent to the ESI probe using the calibration delivery system. An automatic calibration was performed every 5 injections, and all MS and MS/MS calibrant ions were observed to have a mass measurement error
