John Mills (Presenter)
Mayo Clinic
Bio: John Mills received both a B.Sc. and Ph.D. in biochemistry from McGill University in Montreal, Quebec. After his Ph.D., Dr. Mills completed a clinical chemistry post-doctoral fellowship at the Mayo Clinic in Rochester, MN. Currently, Dr. Mills is completing a clinical molecular genetics fellowship at Mayo Clinic. Dr. Mills has a long term goal of transforming how clinical laboratories screen, diagnose and monitor for monoclonal gammopathies by developing more automated, sensitive and affordable assays.
Authorship: John R. Mills, Mindy C. Kohlhagen, Surendra Dasari, Maria A.V. Willrich, David R. Barnidge, and David L. Murray
Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN 55905
| Short Abstract For 50 years electrophoretic separation of serum and urine proteins has played a central role in diagnosing and monitoring of plasma cell disorders. Despite limitations in resolution, sensitivity and the necessity for adjunct methods, protein gel electrophoresis and immunofixation electrophoresis remain front-line tests. A mass spectrometry (MS)-based assay was designed to be automatable, simple, cost-saving, sensitive and applicable to the variety of M-proteins encountered clinically. This assay, MASS-FIX, utilizes the unique molecular mass signatures of the different heavy chain and light chain isotypes in combination with nanobody-immunoenrichment to generate information-rich spectra from which M-proteins can identified, isotyped, quantitated. In this study we compared the performance of MASS-FIX to current gel-based electrophoresis assays. |
Long Abstract
Introduction
Plasma cell disorders (PCDs) are centrally characterized by expansion of clonal plasma cells (PCs), which results in an overabundance of a monoclonal Ig referred to as the monoclonal (M) protein. This M-protein is a surrogate marker of the PC clone, such that diagnosis and management of PCDs depends on accurate identification, characterization and quantitation of M-proteins. Depending on the clinical indication (screening, monitoring, or assessing treatment response) different combinations of serum and urine protein electrophoresis (SPEP and UPEP), serum and urine immunofixation electrophoresis (IFE), serum free light chain (FLC) assays, Ig quantitation and immunoglobulin heavy/light chain (HLC) assay are used to detect and measure M-proteins. The heterogeneity of M-proteins associated with different PCDs and limitations of each assay necessitates the use of multiple tests.
To improve the analytical sensitivity and specificity of detecting M-proteins, we have explored mass spectrometry (MS) based methods to measure M-proteins in serum and urine [1-4]. This technology, known collectively as miRAMM (monoclonal immunoglobulin Rapid Accurate Mass Measurement), is based on the fact that each monoclonal immunoglobulin has a conserved amino acid sequence and therefore a conserved molecular mass that can be measured with high accuracy and sensitivity using MS.
Here we have describe a variant of this technology, termed MASS-FIX, which utilizes isotype-specific nanobody enrichment coupled to matrix assisted laser desportion ionization (MALDI) time-of-flight (TOF) mass spectrometry. This assay is capable of screening, isotyping, measuring heavy-light chain ion ratios and improving quantitation of M-proteins. We will discuss the performance of this assay in comparison to current gel-based methodologies. Furthermore, we will present data suggesting this assay can potentially lead to automated M-protein detection.
Methods
We developed a new MALDI mass spectrometry (MS)-based assay that is simple to perform, automatable, economical, sensitive and applicable to analyze the wide variety of M-proteins encountered clinically. This assay, MASS-FIX, utilizes the unique molecular mass signatures of the different immunoglobulin isotypes in combination with nanobody-immunoenrichment to generate information-rich mass spectra from which M-proteins can be identified, isotyped, and quantitated. We compared the performance of MASS-FIX to current gel-based electrophoresis assays.
Immunoglobulin enrichment was performed using commercially available camelid-derived nanobodies directed against the HC constant domains of IgG, IgA, and IgM or the LC constant domains of k and l immunoglobulins (Thermo Fisher Scientific). Briefly, 10 uL of beads were incubated with 20 uL of serum diluted into 180 uL of PBS for at least 30 minutes at room temperature. Alternatively, 10 uL of beads were mixed with 1 mL of urine and 1 mL of 2X PBS and incubated at RT for 30 minutes. Subsequently, the supernatant was removed and beads were washed 3X with 200 uL of PBS and then 2X with 200 uL of water. Samples were eluted with 80 uL of 5% acetic acid containing 50 mM tris[2-carboxyethyl]phosphine (TCEP).
Samples were spotted onto a 96-well microScout polished steel Bruker target plate (Bruker Daltonics) using the sandwich matrix application method. Each spot was first pre-spotted with 0.6 µL of matrix alpha-cyano-4-hydroxycinnamic acid (10 mg/mL; ACHCA) in 50% acetonitrile (ACN) +0.1% trifluoroacetic acid and allowed to dry. Then 0.6 µL of nanobody eluant (5% acetic acid containing 50 mM TCEP) was applied over the dried matrix. After the sample dried an additional 0.6 uL of ACHCA matrix was layered on top of each spot and allowed to dry. Mass analysis was performed in positive ion mode with summation of 500 laser shots using a MALDI-TOF mass spectrometer (Bruker Microflex LT, Germany). Spectra were generated corresponding to a mass range of 9,000 to 32,000 m/z. Data acquisition for each spectrum took <10 seconds.
Mass spectra were overlaid and these overlaid spectra were used to identify and isotype M-proteins. Mass spectra were also used as a correction factor to improve nephelometry-based quantitation. Software was developed to interpret the mass spectra and aid in interpretation.
Results
MASS-FIX detected 100% of M-proteins that were detectable by both urine and serum protein gel electrophoresis and a majority of those identifiable by IFE (N=88 and 182, respectively). In head-to-head comparison with immunofixation electrophoresis, MASS-FIX was more sensitive and provided the same primary isotype information for ~99% of serum M-proteins (N=154). In comparison to protein electrophoresis, MASS-FIX-corrected quantitation improved accuracy and precision of quantitation of M-proteins <1g/dL (N=53). Between-run and within-run CVs were <20% across all samples analyzed by MASS-FIX with M-protein concentrations >0.04 g/dL. In addition, MASS-FIX could simultaneously measure heavy-light chain ion ratios. Serial monitoring of patients with multiple myeloma post-treatment demonstrated that MASS-FIX provided equivalent quantitative information to either protein electrophoresis or the Hevylite assay (N=15).
Discussion
MASS-FIX can identify and isotype in an assay which is simple, cost-effective, automatable and more sensitive than multiple assays used clinically to obtain the same information. MASS-FIX uses a benchtop linear MALDI-TOF MS platform, the same platform that revolutionized bacterial identification in the clinical microbiology laboratory. The results in this study indicate MASS-FIX provides results in close agreement with current gold-standard methods, yet, MASS-FIX can perform a more diverse set of functions compared to current assays while offering full-automation of pre-analytical immunopurification, mass spectral acquisition and M-protein identification. Importantly, MASS-FIX offers a cost-competitive technology that has the potential to lower the costs of screening, diagnosing and managing PCDs.
References & Acknowledgements:
1. Botz, C.M., et al., Detecting monoclonal light chains in urine: microLC-ESI-Q-TOF mass spectrometry compared to immunofixation electrophoresis. British journal of haematology, 2014. 167(3): p. 437-8.
2. Barnidge, D.R., et al., Using mass spectrometry to monitor monoclonal immunoglobulins in patients with a monoclonal gammopathy. Journal of Proteome Research, 2014. 13(3): p. 1419-27.
3. Mills, J.R., D.R. Barnidge, and D.L. Murray, Detecting monoclonal immunoglobulins in human serum using mass spectrometry. Methods, 2015. 81: p. 56-65.
4. Barnidge D. R., K.T.P., Griffin T.J., Murray D.L., Using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry to detect monoclonal immunoglobuli light chains in serum and urine. Rapid Commun Mass Spectrom, 2015. 29: p. 1-4.
5. Barnidge, D.R., et al., Phenotyping polyclonal kappa and lambda light chain molecular mass distributions in patient serum using mass spectrometry. Journal of Proteome Research, 2014. 13(11): p. 5198-205.
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