Profiling of a high mannose-type N-glycosylated lipase using hydrophilic interaction chromatography-mass spectrometry
Graphical abstract
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,
References (25)
The effect of individual N-glycans on enzyme activity
Bioorg. Med. Chem.
(2009)- et al.
Functional role of N-linked glycosylation in human hepatic lipase: asparagine 56 is important for both enzyme activity and secretion
(1993) - et al.
High-resolution glycoform profiling of intact therapeutic proteins by hydrophilic interaction chromatography-mass spectrometry
Talanta
(2018) - et al.
Hyphenation of size exclusion chromatography to native ion mobility mass spectrometry for the analytical characterization of therapeutic antibodies and related products
J. Chromatogr. B Anal. Technol. Biomed. Life Sci.
(2018) - et al.
Potential of hydrophilic interaction chromatography for the analytical characterization of protein biopharmaceuticals
J. Chromatogr., A
(2016) - et al.
Signal enhancement for gradient reversed-phase high-performance liquid chromatography-electrospray ionization mass spectrometry analysis with trifluoroacetic and other strong acid modifiers by postcolumn addition of propionic acid and isopropanol
J. Am. Soc. Mass Spectrom.
(1995) - et al.
Computer-aided gradient optimization of hydrophilic interaction liquid chromatographic separations of intact proteins and protein glycoforms
J. Chromatogr., A
(2019) - et al.
Analysis of recombinant monoclonal antibodies in hydrophilic interaction chromatography: a generic method development approach
J. Pharmaceut. Biomed. Anal.
(2017) - et al.
Evolution of glycan diversity
- et al.
Implications of cellobiohydrolase glycosylation for use in biomass conversion, Biotechnol
Biofuels
(2008)
Role of N-linked glycosylation in the secretion and enzymatic properties of Rhizopus chinensis lipase expressed in Pichia pastoris
Microb. Cell Factories
Site-directed removal of N-glycosylation sites in human gastric lipase
Eur. J. Biochem.
Cited by (17)
Hydrophilic interaction liquid chromatography promotes the development of bio-separation and bio-analytical chemistry
2023, TrAC - Trends in Analytical ChemistryInfluence of ion-pairing reagents on the separation of intact glycoproteins using hydrophilic-interaction liquid chromatography - high-resolution mass spectrometry
2023, Journal of Chromatography ACitation Excerpt :Trifluoroacetic acid (TFA) is the most widely used IPR for the separation of proteins and specifically for the separation of glycoforms of glycoproteins. It provides high separation efficiency, reduces peak tailing, it facilitates protein solubilization, and improves protein recovery [8]. Nevertheless, the use of TFA has some drawbacks when coupling HILIC to MS. TFA inhibits the ionization of the analytes, causing a low signal, distributed over multiple protein-TFA adducts.
LC-MS/MS in glycomics and glycoproteomics analyses
2021, Carbohydrate Analysis by Modern Liquid Phase Separation TechniquesHydrophilic interaction liquid chromatography-mass spectrometry for the characterization of glycoproteins at the glycan, peptide, subunit, and intact level
2021, Carbohydrate Analysis by Modern Liquid Phase Separation Techniques
- 1
A.F.G. Gargano and O. Schouten are both first authors of the manuscript as they contributed equally to the experimental work, data analysis and preparation of figures. The full list of the author’s contribution is reported in the CRediT author statement.