a) The goal of this study is to develop and characterize mass spectrometry assays for antibody quantification to assess multiple myeloma patients; the results generated from these peptide-based techniques will be compared data generated with gel-based and capillary serum protein electrophoresis (SPEP), which represents the current standard of care.

b) Monoclonal antibody concentrations are currently determined using protein electrophoresis with either serum aliquots (SPEP) or Urine samples (UPEP), the specific class of myeloma is then determined using immunofixation. Liquid chromatography coupled to multiple reaction monitoring mass spectrometry (LC-MRM) assays have been developed to quantify all antibodies (Ig G, A, M, D, E, kappa, and lambda) and their isoforms in serum samples by measuring against known concentrations of stable isotope labeled internal standards using a triple quadrupole mass spectrometer. For assay development, at least two peptides representing each antibody isoform were derived from either LC-MS/MS runs based on excised gamma bands from stored SPEP gels of previous myeloma patients; these experimental results could also be supplemented by in silico workup of the protein sequence (i.e. theoretical peptide predictions). For each peptide target, several transitions (pairs of intact peptide masses and fragment ion masses) are programmed into the triple quadrupole for targeted detection using LC-MRM. Samples are analyzed in triplicate and compared with SPEP results.

c) LC-MRM analysis has been performed on more than 40 serum samples collected from multiple myeloma patients. Using the selected peptides, protein amounts were calculated and compared with previous clinical SPEP results. The panel of antibodies presents unique opportunities and challenges for examining the utility of LC-MRM monitoring; for example, IgD and IgE increases can be detected with LC-MRM well below the 1 mg/ml limit of SPEP (at approximately 20 micrograms/ml). On the other hand, quantification of IgG1 with LC-MRM must be extremely accurate (< 10% CV) to detect the monoclonal protein against the background of that protein normally found in serum. To date, LC-MRM can detect all SPEP positive patients, and this method shows promise in detecting increases in antibody concentrations before they can be detected with SPEP.

d) Quantitative mass spectrometry measurements for the antibodies combine the value of current SPEP and immunofixation experiments. Our results show that peptide-based monitoring can be as effective as protein-based monitoring. In addition, LC-MRM can detect light chain only disease, IgA myeloma that do not present a clear band in the gamma region of SPEP, and oligoclonal or polyclonal disease. These data enable broader commentary on the utility of LC-MRM in clinical protein measurements.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5557.