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High-throughput characterization of increasingly complex and heterogeneous protein structures (including both primary and higher order structures) is now required in a variety of fields ranging from personalized medicine (biomarkers) to industrial-scale production of recombinant proteins (for both product quality control and feedback adaptive process control). However, extensive structural characterization usually involves several multi-step processes that are both time- and labor-consuming, and frequently cannot be implemented in a high-throughput format. Additional complication arises from the presence of multiple protein sub-populations in the analytical\/clinical\/production sample, which may exhibit altered functional or biophysical properties despite having very similar structural characteristics (e.g<\/em>, small soluble aggregates,\u00a0aberrant glycoforms<\/a>, disulfide-scrambled species,\u00a0etc<\/em>.). Our research aims at developing a robust and versatile analytical technology using the novel\u00a0cross-path reactive chromatography (XP-RC) platform<\/a>\u00a0with on-line detection by ESI MS augmented by protein ion manipulation in the gas phase (including both conventional top-down MS\/MS and\u00a0the limited charge reduction technique developed in our laboratory<\/a>). XP-RC allows protein chemical modifications (such as disulfide reduction and covalent labeling) to be combined in-line with the separation step and enables real-time MS measurements that are not adversely affected by components incompatible with the ESI process. This is achieved by utilizing the unique elution characteristics (retention) of proteins and small-molecule reagents in non-denaturing chromatographic media (size exclusion or ion exchange); during their retention the proteins can be exposed to various reagents to induce the desired modification(s) in a highly controlled fashion.<\/p>\n Related publications<\/strong><\/p>\n Y. Yang, D.G. Ivanov, M.D. Levin, B. Olenyuk, O. Cordova-Robles, B. Cederstrom, J.E. Schnitzer, and I.A. Kaltashov. Characterization of large immune complexes with size exclusion chromatography and native mass spectrometry supplemented with gas phase ion chemistry<\/a>. Anal. Chem<\/em>., 2024<\/strong>, 96<\/em>, 2822-2829 (doi: 10.1021\/acs.analchem.3c03278<\/span>)<\/p>\n W. Yang, D.G. Ivanov and I.A. Kaltashov. Extending the capabilities of intact-mass analyses to monoclonal immunoglobulins of the E-isotype (IgE)<\/u><\/a>. MAbs <\/em>2022<\/strong>, 14<\/em>, 2103906 (doi: 10.1080\/19420862.2022.2103906<\/span>)<\/p>\n D.G. Ivanov, Y. Yang, J.W. Pawlowski, I.J. Carrick and I.A. Kaltashov. Rapid evaluation of the extent of haptoglobin glycosylation using orthogonal intact-mass MS approaches and multivariate analysis<\/u><\/a>. Anal. Chem<\/em>. 2022<\/strong>, 94<\/em>, 5140-5148<\/p>\n Y. Yang, D.G. Ivanov and I.A. Kaltashov. The challenge of structural heterogeneity in the native mass spectrometry studies of the SARS-CoV-2 spike protein interactions with its host cell-surface receptor<\/u>. Anal. Bioanal. Chem<\/em>. 2021<\/strong>, 413<\/em>, 7205-7214<\/p>\n Y. Yang, C. Niu, C.E. Bobst and I.A. Kaltashov. Charge manipulation using solution and gas-phase chemistry to facilitate analysis of highly heterogeneous protein complexes in native mass spectrometry<\/a>. Anal. Chem.<\/em> 2021<\/strong>, 93<\/em>, 3337\u20133342<\/p>\n I.A. Kaltashov, J.W. Pawlowski, W. Yang, K. Muneeruddin, H. Yao, C.E. Bobst, and A.N. Lipatnikov. LC\/MS at the whole protein level: Studies of biomolecular structure and interactions using native LC\/MS and cross-path reactive chromatography (XP-RC) MS<\/u>. Methods<\/em>, 2018<\/strong>, 144<\/em>, 14-26<\/p>\n J.W. Pawlowski, I. Carrick and I.A. Kaltashov. Integration of On-column Chemical Reactions in Protein Characterization by LC\/MS: Cross-Path Reactive Chromatography<\/u>. Anal. Chem<\/em>., 2018<\/strong>, 90<\/em>, 1348-1355<\/p>\n K. Muneeruddin, C.E. Bobst, R. Frenkel, D. Houde, I. Turyan, Z. Sosic and I.A. Kaltashov. Characterization of a PEGylated protein therapeutic by ion exchange chromatography with on-line detection by native ESI MS and MS\/MS<\/u>. Analyst<\/em> 2017<\/strong>, 142, 336<\/p>\n R.R. Abzalimov and I.A. Kaltashov. Electrospray ionization mass spectrometry of highly heterogeneous protein systems: protein ion charge state assignment via incomplete charge reduction<\/u>. Anal. Chem<\/em>., 2010<\/strong>, 82<\/em>, 7523-7526<\/p>\n","protected":false},"excerpt":{"rendered":" High-throughput characterization of increasingly complex and heterogeneous protein structures (including both primary and higher order structures) is now required in a variety of fields ranging from personalized medicine (biomarkers) to industrial-scale production of recombinant proteins (for both product quality control and feedback adaptive process control). However, extensive structural characterization usually involves several multi-step processes that … Continue reading Novel Approaches to Characterization of Complex and Highly Heterogeneous Biopolymers<\/span><\/a><\/p>\n","protected":false},"author":69,"featured_media":0,"parent":0,"menu_order":0,"comment_status":"closed","ping_status":"closed","template":"","meta":{"footnotes":""},"class_list":["post-356","page","type-page","status-publish","hentry"],"_links":{"self":[{"href":"https:\/\/elements.chem.umass.edu\/kaltashovlab\/wp-json\/wp\/v2\/pages\/356","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/elements.chem.umass.edu\/kaltashovlab\/wp-json\/wp\/v2\/pages"}],"about":[{"href":"https:\/\/elements.chem.umass.edu\/kaltashovlab\/wp-json\/wp\/v2\/types\/page"}],"author":[{"embeddable":true,"href":"https:\/\/elements.chem.umass.edu\/kaltashovlab\/wp-json\/wp\/v2\/users\/69"}],"replies":[{"embeddable":true,"href":"https:\/\/elements.chem.umass.edu\/kaltashovlab\/wp-json\/wp\/v2\/comments?post=356"}],"version-history":[{"count":9,"href":"https:\/\/elements.chem.umass.edu\/kaltashovlab\/wp-json\/wp\/v2\/pages\/356\/revisions"}],"predecessor-version":[{"id":1519,"href":"https:\/\/elements.chem.umass.edu\/kaltashovlab\/wp-json\/wp\/v2\/pages\/356\/revisions\/1519"}],"wp:attachment":[{"href":"https:\/\/elements.chem.umass.edu\/kaltashovlab\/wp-json\/wp\/v2\/media?parent=356"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}