MSACL 2018 US Abstract

Topic: Proteomics

The First Precision Diagnosis of Multiple Myeloma by Use of 21 Tesla FT-ICR Top/Middle-Down de novo Sequencing

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 (4), Surendra Dasari (4), Angela Dispenzieri (4), Christopher L Hendrickson (1,2), and Alan G. Marshall(1,2)
(1) Florida State University, Tallahassee, FL (2) National High Magnetic Field Laboratory, Tallahassee, FL (3) The Binding Site Inc., Rochester, MN (4) Mayo Clinic, Rochester, MN

Short Abstract

Multiple myeloma is a B cell malignancy characterized by a plasma cell clonal expansion. If there is clinical suspicion of a multiple myeloma, serum is tested for the presence of elevated levels of a monoclonal immunoglobulin (mIg) secreted by clonal plasma cells. We describe the first top/middle-down de novo sequencing of mIg in serum with the advantages of ultrahigh mass accuracy and extensive residue cleavages. There may be certain predilections for disease type based on immunoglobulin gene usage, and typical cloning experiments are expensive, invasive, and laborious. Herein we report a non-invasive method for sequencing mIg proteins from the blood.

Long Abstract

Introduction

Multiple myeloma is a term used to describe a B cell malignancy characterized by a plasma cell clonal expansion. If there is clinical suspicion of a multiple myeloma, serum and urine are tested for the presence of elevated levels of a monoclonal immunoglobulin (mIg) secreted by clonal plasma cells [1]. We describe top-down and middle-down de novo sequencing of mIgs from patients with multiple myeloma 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 mIgs, and sets a benchmark for top-down MS/MS analysis of multiple mIgs in serum. To the best of our knowledge, this report is the first multiple myeloma precision diagnosis by top/middle-down de novo sequencing. In addition, there may be certain predilections for disease type based on immunoglobulin gene usage, and typical cloning experiments are expensive, invasive, and laborious. Herein we report a non-invasive method for sequencing mIg proteins from the blood.

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 5 mM 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 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 [3]) and collisional induced dissociation (CID). Data were manually interpreted with Xcalibur 2.1 software (Thermo). Antibody constant region sequence coverage was determined by Xtract deconvolution and fragments matched to the putative sequence by use of ProSight Lite (10 ppm fragment mass tolerance) [4]. Antibody variable region sequence characterization was performed by an in-house program, “Amino Acid Finder”.

Results

Prior work focused on monitoring patient sera for a monoclonal immunoglobulin by use of a Q-TOF MS platform [5]. However, we recently demonstrated that LC-MS/MS based on our FT-ICR MS provides better mass measurement accuracy and greater sequence coverage for isolated monoclonal immunoglobulin light and heavy chains [6]. The ultrahigh mass resolution yields 119 isotopic peaks with a combined rms error of 0.2-0.4 ppm for antibody light chain, Fc/2, and Fd subunits with sequence coverages of 82%, 80%, and 75%. We further applied this method to analyze the mIgs from multiple myeloma patient serum (our group was blind to the M protein gene sequencing result). Based on our in-house developed top/middle-down de novo sequencing software, the mIg light chain was assigned to kappa IGKV1-33 germline sequence with seven amino acid mutations. The assigned sequence was compared to the bone marrow DNA sequencing result (converted to amino acids), and 100% matching of assigned residues was achieved. This method performance evaluation suggests that the nanoLC FT-ICR MS/MS can be used to complement current genome sequencing approaches as a less invasive method. In addition, this new approach may avoid false-negatives from bone marrow aspiration (for genome sequencing).

In another clinical sample, two different mIgs light chains were detected. After de novo sequencing with 21 tesla FT-ICR MS/MS and our in-house developed software, the two light chains belong to different light chain germline sequence kappa IGKV3-11 and kappa IGKV1-16, indicating that our approach can simultaneously characterize more than one mAb light chain corresponding to potential disease progression (e.g., malignant plasma cells mutation). In addition, the heavy chain variable region was determined to be the VH3-9 germline sequence. The IGKV1-33 and IGKV1-16 germline sequences from the above two patients belong to the kappa I category, which is prone to AL amyloidosis. And post-translational modifications (heavy chain glycoforms) from multiple myeloma samples are well characterized and differ from the glycoforms in healthy human samples.

Conclusions & Discussion

The goal of this work was to evaluate the performance of our nano-LC 21 T FT-ICR MS/MS instrument for characterizing an mIg in human serum, in the hope of establishing a protocol for utilizing this unique instrumentation in situations for which mass spectrometers currently used in clinical laboratories would not suffice. Our findings demonstrate the widespread capabilities of this instrument platform, as evidenced by the outstanding mass measurement accuracy and extensive top-down MS/MS fragmentation data, enabling confident characterization of unknown mIgs in the presence of a polyclonal background. The results shown here serve as a blueprint for future characterization of endogenous mIgs 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 de novo characterization of complete mIg sequences in patients with multiple myeloma. The present method will enable better understanding of the pathogenesis of multiple myeloma and potentially guide personalized therapy. Preliminary results indicate that top-down and middle-down analysis of an unknown mIg in serum is achievable from a few LC-MS/MS experiments.


References & Acknowledgements:

Work supported by the National Science Foundation through DMR-1157490 and the State of Florida.

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. 2013, 19, 985-994.

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. 2015, 26, 1626-1632.

3. Weisbrod, C. R.; Kaiser, N. K.; Syka, J. E. P.; Early, D.; Mullen, C.; Dunyach, J.-J.; English, A. M.; Anderson, L. C.; Blakney, G. T.; Shabanowitz, J.; Hendrickson C. L.; Marshall, A. G.; Hunt, D. F. Front-End Electron Transfer Dissociation Coupled to a 21 Tesla FT-ICR Mass Spectrometer for Intact Protein Sequence Analysis, J. Am. Soc. Mass Spectrom, 2017, 28, 1787-1795.

4. 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. 2015, 15, 1235-1238.

5. 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. 2014, 13, 1419-1427.

6. He, L., Anderson, L.C., Barnidge, D.R., Murray, D.L., Hendrickson, C.L., Marshall, A.G.: Analysis of Monoclonal Antibodies in Human Serum as a Model for Clinical Monoclonal Gammopathy by Use of 21 Tesla FT-ICR Top-Down and Middle-Down MS/MS. J. Am. Soc. Mass Spectrom. 2017, 28, 827-838.


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