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Cracking proteoform complexity of ovalbumin with anion exchange chromatography-high resolution mass spectrometry under native conditions Florian Fussl, Angela Criscuolo, Ken Cook, Kai Scheffler, and Jonathan Bones J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.9b00375 • Publication Date (Web): 03 Sep 2019 Downloaded from pubs.acs.org on September 3, 2019
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Journal of Proteome Research
Cracking proteoform complexity of ovalbumin with anion exchange chromatography-high resolution mass spectrometry under native conditions
Florian Füssl1, Angela Criscuolo2,3,4, Ken Cook5, Kai Scheffler6, Jonathan Bones1,7,*
1NIBRT
– The National Institute for Bioprocessing Research and Training, Foster Avenue,
Mount Merrion, Blackrock, Co. Dublin, A94 X099, Ireland 2Institute
of Bioanalytical Chemistry, Faculty of Chemistry and Mineralogy, Universität
Leipzig, Germany 3Center
for Biotechnology and Biomedicine, Universität Leipzig, Germany
4Thermo
Fisher Scientific, Hanna-Kunath-Strasse 11, 28199 Bremen, Germany
5Thermo
Fisher Scientific, Stafford House, 1 Boundary Park, Hemel Hempstead, HP2 7GE,
United Kingdom 6Thermo
7School
Fisher Scientific, Dornierstrasse 4, 82110 Germering, Germany
of Chemical and Bioprocess Engineering, University College Dublin, Belfield,
Dublin
*Correspondence: Email:
[email protected], tel: +35312158100, fax: +35312158116.
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Abstract Posttranslational modifications of proteins play fundamental roles in protein function in health and disease. More than 600 different types of posttranslational modifications are known, many of them being of extremely low abundance, causing subtle changes in physicochemical properties and posing an extreme challenge to analytical methods required for their characterisation. Here, we report the development of a novel pH gradient based anion exchange chromatography method which can be directly interfaced to Orbitrap-based mass spectrometry for the comprehensive characterisation of proteoforms on the intact protein level under native conditions. The analysis of four different proteins demonstrates outstanding chromatographic selectivity while the mass spectra obtained are of excellent quality enabling the identification of proteoforms, including near isobaric variants, spanning four orders of magnitude in abundance. An in-depth analysis of ovalbumin from chicken egg white yielded the identification and relative quantification of more than 150 different proteoforms including fragmented and dimeric forms. More than 20 different ovalbumin charge variants together with their glycoform distributions were identified and quantified, many of which have not been previously reported.
Keywords anion exchange chromatography, high resolution mass spectrometry, native mass spectrometry, AEXMS, charge variant analysis, pH-gradient, proteoform, anionic protein, ovalbumin, posttranslational modification,
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Journal of Proteome Research
Introduction With more than 200 different types and a myriad of genes devoted to their regulation, posttranslational modifications (PTMs) of proteins are fundamental for the persistence of complex life1-2. PTMs can strongly alter the properties and functions of proteins and the presence of specific posttranslational modifications is reflective of the physiological state of a biological system3. For exogenous proteins like biopharmaceuticals produced using recombinant methods, posttranslational modifications represent critical product quality attributes, which may impact drug potency and patient safety, as has been shown for monoclonal antibodies (mAbs)4-8. Charge-sensitive separation modes like ion exchange chromatography (IEC), capillary electrophoresis (CE) and capillary isoelectric focusing (cIEF) are routinely used for the separation and characterisation of proteoforms, due to their high selectivity, allowing for the separation of protein variants with minimal difference in their properties, i.e. net surface charge. However, charge-based separation methods are generally not ideally suited for coupling to mass spectrometry (MS) due to non-compatibility of the buffer systems or background electrolytes used to facilitate the separation9-12. Interfacing of these methods to MS is highly desired, generating analytical synergy from the combination of the excellent separation selectivity with the potential for confident, molecular mass based identification and quantitation. Consequently, a number of reports have appeared in recent years to link charge mediated separation chemistries with mass spectrometric detection 9, 13-20. Several attempts have been made to directly couple cation exchange chromatography (CEX) of intact therapeutic proteins to MS detection, resulting in generically applicable methods for the screening of mAb charge variants 10, 21-25. Notably, the majority of research undertaken thus far has focused on the establishment of methods for the analysis of cationic proteins, even though most proteins present in biological systems are anionic26. This focus on cationic proteins has been driven by the desire to have methods to characterise mAbs, as the vast majority of these pharmaceutically relevant molecules exhibit isoelectric points (pI) in the basic region making them amenable to charge variant separation using CEX. Recently, a method describing on-line anion exchange chromatography (AEX) coupled to MS for the separation of human serum albumin (HSA) proteoforms was reported that utilised a volatile salt
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gradient and time of flight (ToF) mass spectrometry resulting in the identification of nine different HSA proteoforms27. Here, we present a novel method for on-line AEX-MS of intact proteins based on pH gradient elution and highly sensitive Orbitrap mass spectrometric detection under native conditions. pH gradient elution enables the use of low ionic strength buffers capable of generating a linear pH gradient, that facilitate excellent chromatographic selectivity and generic applicability for anionic proteins with pI values
