Raj A. Parikh, Lohra M. Miller, Benjamin E. Draper, Lavelay Kizekai, Balasubrahmanyam Addepalli, Michelle Chen, Matthew A. Lauber* and Martin F. Jarrold*,
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Here, we show how CD-MS, leveraging high-throughput methods such as multiple ion charge extraction (MICE), can match the time scale of SEC, extending the samples amenable to separation analysis by SEC-CD-MS into the megadalton regime and beyond. As part of this work, we have developed low flow ultrawidepore (1000 Å pore size) SEC using narrow bore columns to optimize the coupling between SEC and CD-MS. The analysis of monoclonal antibodies, thyroglobulin, bacteriophage Qβ virus-like particles (VLPs), and hepatitis B virus VLPs, showcases the capabilities of SEC-CD-MS over a broad mass range including the high mass range previously inaccessible for online separation with MS. These findings are complemented by a parallel study using multiangle light scattering (SEC-MALS). SEC-CD-MS and SEC-MALS provide complementary information that is valuable for characterization of complex biologics and nanoparticles. Finally, our results open the door to integration of high throughput CD-MS with other separation techniques for both large and small macromolecules.</p>","PeriodicalId":27,"journal":{"name":"Analytical Chemistry","volume":"97 5","pages":"3036–3044 3036–3044"},"PeriodicalIF":6.7000,"publicationDate":"2025-01-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.acs.org/doi/epdf/10.1021/acs.analchem.4c06084","citationCount":"0","resultStr":"{\"title\":\"Coupling of Size Exclusion Chromatography to High Throughput Charge Detection Mass Spectrometry for the Analysis of Large Proteins and Virus-like Particles\",\"authors\":\"Raj A. Parikh, Lohra M. Miller, Benjamin E. Draper, Lavelay Kizekai, Balasubrahmanyam Addepalli, Michelle Chen, Matthew A. Lauber* and Martin F. 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As part of this work, we have developed low flow ultrawidepore (1000 Å pore size) SEC using narrow bore columns to optimize the coupling between SEC and CD-MS. The analysis of monoclonal antibodies, thyroglobulin, bacteriophage Qβ virus-like particles (VLPs), and hepatitis B virus VLPs, showcases the capabilities of SEC-CD-MS over a broad mass range including the high mass range previously inaccessible for online separation with MS. These findings are complemented by a parallel study using multiangle light scattering (SEC-MALS). SEC-CD-MS and SEC-MALS provide complementary information that is valuable for characterization of complex biologics and nanoparticles. 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Coupling of Size Exclusion Chromatography to High Throughput Charge Detection Mass Spectrometry for the Analysis of Large Proteins and Virus-like Particles
Charge detection mass spectrometry (CD-MS) is an emerging single-particle technique where both the m/z and charge are measured individually to determine each ion’s mass. It is particularly well-suited for analyzing high mass and heterogeneous samples. With conventional MS, the loss of charge state resolution with high mass samples has hindered the direct coupling of MS to separation techniques like size exclusion chromatography (SEC) and forced the use of lower resolution detectors. Here, we show how CD-MS, leveraging high-throughput methods such as multiple ion charge extraction (MICE), can match the time scale of SEC, extending the samples amenable to separation analysis by SEC-CD-MS into the megadalton regime and beyond. As part of this work, we have developed low flow ultrawidepore (1000 Å pore size) SEC using narrow bore columns to optimize the coupling between SEC and CD-MS. The analysis of monoclonal antibodies, thyroglobulin, bacteriophage Qβ virus-like particles (VLPs), and hepatitis B virus VLPs, showcases the capabilities of SEC-CD-MS over a broad mass range including the high mass range previously inaccessible for online separation with MS. These findings are complemented by a parallel study using multiangle light scattering (SEC-MALS). SEC-CD-MS and SEC-MALS provide complementary information that is valuable for characterization of complex biologics and nanoparticles. Finally, our results open the door to integration of high throughput CD-MS with other separation techniques for both large and small macromolecules.
期刊介绍:
Analytical Chemistry, a peer-reviewed research journal, focuses on disseminating new and original knowledge across all branches of analytical chemistry. Fundamental articles may explore general principles of chemical measurement science and need not directly address existing or potential analytical methodology. They can be entirely theoretical or report experimental results. Contributions may cover various phases of analytical operations, including sampling, bioanalysis, electrochemistry, mass spectrometry, microscale and nanoscale systems, environmental analysis, separations, spectroscopy, chemical reactions and selectivity, instrumentation, imaging, surface analysis, and data processing. Papers discussing known analytical methods should present a significant, original application of the method, a notable improvement, or results on an important analyte.