Lidong He (Presenter)
Florida State University
Bio: Lidong He is a graduate student at National High Magnetic Field Laboratory and Florida State University. His interest is FT-ICR MS applications in proteomics and metabolomics for clinical diagnosis.
Authorship: Lidong He(1), Lissa C. Anderson(2), David R. Barnidge(3), David L. Murray(3), Christopher L Hendrickson(1,2), and Alan G. Marshall(1,2)
(1) Florida State University, Tallahassee, FL (2) Ion Cyclotron Resonance Program, National High Magnetic Field Laboratory, Tallahassee, FL (3) Mayo Clinic, Rochester, MN
| Short Abstract Monoclonal gammopathy is a B cell proliferative disorder characterized by a plasma cell clonal expansion. If there is clinical suspicion of a monoclonal gammopathy, serum and urine are tested for the presence of elevated levels of a monoclonal immunoglobulin secreted by clonal plasma cells. Here, we describe top-down and middle-down analysis of immunoglobulins in serum with the advantages of fast sample preparation, ultrahigh mass accuracy, and extensive residue cleavages by use of 21 tesla FT-ICR MS/MS. To our knowledge, this is the first time that extensive top-down cleavages for both variable and constant regions have been achieved for mAbs in a human serum background. |
Long Abstract
Introduction:
Monoclonal gammopathy is a term used to describe a B cell proliferative disorder characterized by a plasma cell clonal expansion. If there is clinical suspicion of a monoclonal gammopathy such as multiple myeloma, serum and urine are tested for the presence of elevated levels of a monoclonal immunoglobulin secreted by clonal plasma cells [1]. Here, we describe top-down and middle-down analysis of immunoglobulins with the advantages of fast sample preparation with minimal artifacts, ultrahigh mass accuracy, and extensive residue cleavages by use of 21 tesla FT-ICR MS/MS. Nano-LC 21 T FT-ICR MS/MS provides nonpareil mass resolution, mass accuracy, and sequence coverage for mAbs, and sets a benchmark for top-down MS/MS analysis of multiple mAbs in serum. To the best of our knowledge, this is the first time that extensive top-down cleavages for both variable and constant regions have been achieved for mAbs in a human serum background.
Methods:
Immunoglobulins were purified from human serum by use of Melon Gel (Thermo Fisher Scientific, Waltham, MA) and further cleaned with an Amicon Ultra-0.5 Centrifugal Filter Unit with Ultracel-10 membrane (EMD Millipore, Darmstadt, Germany). Disulfide bonds were then reduced with 0.1 M TCEP in 0.1% acetic acid at 75 °C for 15 min to produce intact antibody light (~24 kDa) and heavy (~50 kDa) chains. For pure monoclonal immunoglobulin analysis, adalimumab was reduced with TCEP directly. For immunoglobulin IdeS digestion and reduction, one unit of IdeS (FabRICATOR, Genovis, Cambridge, MA) was added per microgram of IgG and incubated at 37 °C for 30 min as instructed by the manufacturer. Following digestion, samples were reduced with TCEP and acidified with 1% acetic acid to pH ~2 prior to nano-LC ESI MS/MS analysis. Mass spectra were acquired with our custom-built 21 T FT-ICR mass spectrometer [2], and MS/MS experiments were conducted with both electron transfer dissociation (ETD) and collisional induced dissociation (CID). Data were manually interpreted with Xcalibur 2.1 software (Thermo), and sequence coverage determined by Xtract deconvolution, and fragments were matched to the putative sequence by use of ProSight Lite [3].
Results and Conclusions:
Prior work focused on monitoring patient sera for a monoclonal immunoglobulin by use of a Q-TOF MS platform [4]. However, we recently demonstrated that our FT-ICR based LC-MS/MS provides better mass measurement accuracy and greater sequence coverage for isolated monoclonal immunoglobulin light and heavy chains [5]. The ultrahigh mass accuracy yields 119 isotopic peaks with a combined rms error 0.2-0.4 ppm for antibody light chain, Fc/2, and Fd subunits with sequence coverages of 82%, 80%, and 75%. Extension to a monoclonal antibody in human serum as a monoclonal gammopathy model yielded 58% sequence coverage from two nano-LC MS/MS runs. Using adalimumab as a model for high-abundance endogenous monoclonal immunoglobulin, we demonstrate that the light chain, heavy chain, and heavy chain IdeS-generated fragments can be extensively characterized, including N-glycosylation site localization, and three heavy chain isoforms (K0G0F, K0G1F, and K1G0F). A blind analysis of five unknown monoclonal antibodies at clinically relevant concentrations in human serum resulted in correct identification of all five unknowns. We further extended the method to demonstrate the ability to quantify the intact light chain from a mAb in a polyclonal background matrix as a model for a patient with a monoclonal gammopathy—a first for our nano-LC 21 T FT-ICR MS/MS instrument platform. Preliminary results of samples from patients with monoclonal gammopathy have been obtained.
The goal of this work was to evaluate the performance of our nano-LC 21 T FT-ICR MS/MS instrument for characterizing a mAb in human serum, in the hope of establishing a protocol for utilizing this unique instrumentation in situations for which mass spectrometers used in clinical laboratories would not suffice. Our findings demonstrate the widespread capabilities of this instrument platform, as evidence by the outstanding mass measurement accuracy and extensive top-down MS/MS fragmentation data, enabling confident assignment of unknown mAbs in the presence of a polyclonal background. The results shown here serve as a blueprint for future characterization of endogenous mAbs in patients with a variety of immune system disorders that cannot be achieved by gene sequencing alone. The results also represent a successful union of clinical laboratory expertise with a national analytical resource.
We shall report progress toward characterization of monoclonal immunoglobulins in patients with monoclonal gammopathy. Preliminary results indicate that top-down and middle-down analysis of unknown mAb in serum is achievable in a few LC-MS/MS experiments.
References & Acknowledgements:
References
1.Agarwal, A., Ghobrial, I.M.: Monoclonal gammopathy of undetermined significance and smoldering multiple myeloma: a review of the current understanding of epidemiology, biology, risk stratification, and management of myeloma precursor disease. Clin. Cancer Res. 19, 985-994 (2013)
2.Hendrickson, C.L.; Quinn, J.P., Kaiser, N.K., Smith, D.F., Blakney, G.T., Chen, T., Marshall, A.G., Weisbrod, C.R., Beu, S.C.: 21 Tesla Fourier Transform Ion Cyclotron Resonance Mass Spectrometer: A National Resource for Ultrahigh Resolution Mass Analysis. J. Am. Soc. Mass Spectrom. 26, 1626-1632 (2015)
3.Fellers, R.T., Greer, J.B., Early, B.P., Yu, X., LeDuc, R.D., Kelleher, N.L., Thomas, P.M.: ProSight Lite: graphical software to analyze top-down mass spectrometry data. Proteomics. 15, 1235-1238 (2015)
4.Barnidge, D.R., Dasari, S., Botz, C.M., Murray, D.H., Snyder, M.R., Katzmann, J.A., Dispenzieri, A., Murray, D.L.: Using Mass Spectrometry to Monitor Monoclonal Immunoglobulins in Patients with a Monoclonal Gammopathy. J. Proteome Res. 13, 1419-1427 (2014)
5.He, L.; Anderson, L.C.; Barnidge, D.R.; Murray, D.L.; Hendrickson, C.L.; Marshall, A.G. “Analysis of Monoclonal Immunoglobulins from Multiple Myeloma Patients by Use of 21 Tesla FT-ICR MS/MS,” 64th American Society for Mass Spectrometry and Allied Topics, San Antonio, TX, June 5-9, 2016. Poster MP607.
Acknowledgments
Work supported by the National Science Foundation through DMR-1157490 and the State of Florida.
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