Profiling of a high mannose-type N-glycosylated lipase using hydrophilic interaction chromatography-mass spectrometry

A. F.G. Gargano*, O. Schouten, G. van Schaick, L. S. Roca, J. H. van den Berg-Verleg, R. Haselberg, M. Akeroyd, N. Abello, G. W. Somsen

*Corresponding author for this work

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Many industrial enzymes exhibit macro- and micro-heterogeneity due to co-occurring post-translational modifications. The resulting proteoforms may have different activity and stability and, therefore, the characterization of their distributions is of interest in the development and monitoring of enzyme products. Protein glycosylation may play a critical role as it can influence the expression, physical and biochemical properties of an enzyme. We report the use of hydrophilic interaction liquid chromatography-mass spectrometry (HILIC-MS) to profile intact glycoform distributions of high mannose-type N-glycosylated proteins, using an industrially produced fungal lipase for the food industry as an example. We compared these results with conventional reversed phase LC-MS (RPLC-MS) and sodium dodecyl sulfate–polyacrylamide gel-electrophoresis (SDS-PAGE). HILIC appeared superior in resolving lipase heterogeneity, facilitating mass assignment of N-glycoforms and sequence variants. In order to understand the glycoform selectivity provided by HILIC, fractions from the four main HILIC elution bands for lipase were taken and subjected to SDS-PAGE and bottom-up proteomic analysis. These analyses enabled the identification of the most abundant glycosylation sites present in each fraction and corroborated the capacity of HILIC to separate protein glycoforms based on the number of glycosylation sites occupied. Compared to RPLC-MS, HILIC-MS reducted the sample complexity delivered to the mass spectrometer, facilitating the assignment of the masses of glycoforms and sequence variants as well as increasing the number of glycoforms detected (69 more proteoforms, 177% increase). The HILIC-MS method required relatively short analysis time (<30 min), in which over 100 glycoforms were distinguished. We suggest that HILIC(-MS) can be a valuable tool in characterizing bioengineering processes aimed at steering protein glycoform expression as well as to check the consistency of product batches.

Original languageEnglish
Pages (from-to)69-77
Number of pages9
JournalAnalytica Chimica Acta
Early online date28 Feb 2020
Publication statusPublished - 1 May 2020


AG acknowledges financial support by the Netherlands Organization for Scientific Research, NWO Veni grant IPA ( 722.015.009 ). LR acknowledges the STAMP project, which is funded under the Horizon 2020 – Excellent Science – European Research Council (ERC) , Project 694151 . The sole responsibility of this publication lies with the authors. The European Union is not responsible for any use that may be made of the information contained therein. The authors would like to thank Peter J. Schoenmakers, and Eva M. Johansson (University of Amsterdam) for their help and valuable discussions.

FundersFunder number
Excellent Science
Netherlands Organization for Scientific Research
Horizon 2020 Framework Programme694151
European Commission
European Research Council
Nederlandse Organisatie voor Wetenschappelijk Onderzoek722.015.009
Horizon 2020


    • High mannose-type N-Glycosylation
    • High-resolution mass spectrometry
    • HILIC
    • Intact protein analysis


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