Characterization of oligosaccharides in recombinant tissue plasminogen activator produced in Chinese hamster ovary cells: two decades of analytical technology development

Analytical Chemistry
Oleg V BorisovReed J Harris

Abstract

Recombinant tissue plasminogen activator (rt-PA) is a well-characterized glycoprotein with a great deal of published information on its structure, post-translational modifications, and O- and N-glycosylation. Most of the characterization was accomplished in the late 1980s. During the past 2 decades, however, mass spectrometry has made a quantum leap forward offering new capabilities in soft electrospray ionization, speed, resolution, and accuracy of mass measurements. From this point of view, it is worthwhile to revisit the characterization of familiar proteins, such as rt-PA, using the new capabilities of modern analytical technology. In this work, we applied LC-MS with state-of-the-art instrumentation to the characterization of glycoforms of rt-PA. This method takes advantage of accurate mass measurements along with a fast "in-source" voltage switching for the detection of characteristic oxonium ions of saccharides. This method confirmed previously identified glycan structures based on existing knowledge of rt-PA glycans. In addition, we identified two novel glycan structures in rt-PA. A low level of Asn142 N-glycosylation was detected at an atypical Asn-Xaa-Cys consensus motif. It was found to be modified predominantly by bi...Continue Reading

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Citations

Mar 28, 2013·Chemical Reviews·William R AlleyMilos V Novotny
Dec 3, 2010·Glycobiology·Takeshi MatsuiKo Kato
Nov 4, 2015·Frontiers in Cellular Neuroscience·Arnaud ChevilleyDenis Vivien
Nov 9, 2010·Biotechnology Journal·Mahdi HemayatkarNoushin Davoudi
Aug 7, 2013·Biotechnology Advances·Vaibhav JadhavNicole Borth
Jul 28, 2016·Journal of Pharmaceutical and Biomedical Analysis·Sachio YamamotoShigeo Suzuki
May 1, 2010·Biomolecular Concepts·Carmen Jiménez-CastellsRicardo Gutiérrez-Gallego
Jan 17, 2017·Analytical and Bioanalytical Chemistry·Kenichiro FurukiKazutoshi Ban

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