Elsevier

Analytica Chimica Acta

Volume 1109, 1 May 2020, Pages 69-77
Analytica Chimica Acta

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

https://doi.org/10.1016/j.aca.2020.02.042Get rights and content

Highlights

  • Comparison of SDS-PAGE, RPLC-MS and HILIC-MS of intact lipase.

  • HILIC-MS allows for high-resolution separation of lipase glycoforms.

  • In-depth glycoform characterization combining bottom-up and intact-protein methods.

Abstract

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.

Introduction

Glycosylation is one of the most common post-translational modifications (PTMs) of proteins. This PTM is generated by the covalent attachment of oligosaccharides (glycans) to the amino acid backbone of a protein, in particular to serine/threonine (O-glycosylation) or asparagine (N-glycosylation) residues. Depending on the type of organism, glycans are processed differently, giving rise to different N-glycosylation. In yeast and fungi, the high-mannose type (varying in the number of neutral mannose units) is dominant, whereas in vertebrates, complex types (including a variety of sugar units like sialic acid) are most prominent [1].

The evidence of the importance of N-glycosylation in regulating glycoprotein expression, structure and function is overwhelming [2]. N-glycosylation has shown to affect several enzyme properties, such as functional activity, conformation, stability and solubility [3]. For example, the partial or complete removal of the N-glycans in lipases resulted in reduced thermal stability and altered activity [2,4,5]. Glycosylation at N308 of human gastric lipase (one of its four N-glycosylation sites) appeared essential for full enzyme activity. Deglycosylated lipase was 50% less active than the wild-type enzyme. Interestingly the lack of glycosylation on the remaining glycosylation sites did not affect enzyme activity [6].

Therefore, profiling the glycosylation of enzymes such as lipases used in e.g. industrial food applications is highly relevant during the development and optimization of the enzyme production. Glycoproteins may be characterized at multiple levels using separation methods coupled to mass spectrometry (MS). Typically the analytical approaches used target either the glycans released from a protein [7], the glycopeptides [8] or the intact glycoprotein [9]. For the determination of the glycoforms expressed, the analysis of intact glycoproteins is the one conserving the highest amount of information but also the more difficult from an analytical chemistry perspective [10].

Several liquid-based hyphenated methods for glycoform profiling of intact proteins have been described, including capillary electrophoresis (CE) and liquid chromatography (LC) [9,11,12]. Coupling separation methods with high-resolution mass spectrometry (MS) enables to resolve the protein heterogeneity according to chemical differences (e.g. charge, hydrophobicity etc.) and measures the accurate masses of the proteoforms.

To date, RPLC, HILIC, IEC, SEC, HIC and CE methods have been applied to the online MS characterization of intact proteins (e.g. Refs. [[13], [14], [15], [16], [17], [18]]). RPLC-based methods typically use a solvent combination that ensures compatibility with electrospray ionization MS and are therefore the most commonly used. However, these methods separate proteins mainly according to the amino acid composition and are typically not sufficiently selective to resolve glycoproteoforms. Recently, HILIC-MS has emerged as an analytical tool for protein analysis with complementary selectivity to RPLC-MS and capable of high-resolution separation of glycoforms [10,11,14,19,20]. HILIC methods for the characterization of glycoproteins are often using neutral stationary phases (e.g. using amide phases). Acetonitrile-water gradients are used with mobile phase additives allowing ion-pair formation with basic protein residues at low pH. As a result, ionic interactions of the protein with the stationary phase are minimized, leaving hydrophilic partitioning and hydrogen bonding as driving forces of retention. Under these conditions, the (neutral) sugars of protein glycans contribute substantially to retention, providing glycoform resolution according to the overall glycan size and composition [10,11,19]. Therefore, HILIC glycoform separations are complementary with respect to capillary electrophoresis (CE) where glycoforms are resolved on the basis of charge differences (e.g. differences in the number of sialic acid) [10].

In our investigation, we used a heavily N-glycosylated lipase as an example of profiling glycoforms with HILIC-MS. The high mannose-type glycosylation on this enzyme is particularly challenging to profile since it has high heterogeneity only based on neutral sugar. We adopted several analytical strategies to characterize this lipase, including intact protein approaches (SDS-PAGE, RPLC-MS and HILIC-MS) and a study of the N-glycosylation by protein digestion. HILIC demonstrated excellent resolving power for glycoforms of the enzyme (varying in the number of neutral sugar units) enabling an in-depth characterization of the proteoforms expressed.

Section snippets

Chemicals

The lipase enzyme studied was provided by DSM Biotechnology Center as a formulation in wheat flour. The enzymes used in the processing described below were PNGase F, Trypsin Gold and EndoHf (Promega). M12 was used as molecular weight ladder (Invitrogen). All solvents and reagents were bought through Sigma Aldrich and of the highest quality available. Deionized water (18.2 MΩ) was obtained from a Milli-Q purification system.

Sample preparation

Lipase sample: 13.50 mg was weighed to which 1.35 mL Milli-Q water

Results and discussion

This study aimed to profile glycoforms of a heavily N-glycosylated lipase protein. A commercially available lipase produced in Aspergillus niger, having a theoretical molecular weight in its mature form of about 28 kDa, was used as a proof of principle. This enzyme is a glycoprotein with four possible N-glycosylation sites (Fig. 1a) and four disulfide bridges (for amino acid sequence, see section S1 of the supporting information). It is highly heterogeneous due to variation in size and position

Conclusions

The detailed characterization of highly glycosylated proteins, such as industrial enzymes and biopharmaceutical products is essential, but challenging. Intact proteins analysis methods using RPLC-MS have limited resolving power for protein glycoforms, potentially leaving a significant number of them undetected.

This study shows that HILIC offers more detailed information on intact protein heterogeneity due to its unique selectivity towards glycoforms. When combined with MS, HILIC significantly

CRediT authorship contribution statement

A.F.G. Gargano: Conceptualization, Methodology, Formal analysis, Investigation, Data curation, Writing - original draft, Writing - review & editing, Visualization, Supervision, Funding acquisition, Project administration. O. Schouten: Resources, Visualization, Formal analysis, Investigation. G. van Schaick: Investigation, Visualization. L.S. Roca: Investigation. J.H. van den Berg-Verleg: Investigation, Visualization. R. Haselberg: Conceptualization, Writing - review & editing, Resources. M.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

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,

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